Microorganisms genetically engineered to have modified N-glycosylation activity

ABSTRACT

Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/062,469, filed Apr. 3, 2008, now U.S. Pat. No. 8,026,083, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/940,212,filed May 25, 2007, and which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/909,904, filed Apr. 3, 2007. The disclosures ofthe prior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

TECHNICAL FIELD

The invention relates to methods of obtaining glycosylated molecules,particularly protein and lipid molecules.

BACKGROUND

High performance expression systems are required to produce mostbiopharmaceuticals (e.g., recombinant proteins) currently underdevelopment. The biological activity of many of these biopharmaceuticalsis dependent on their modification (e.g., phosphorylation orglycosylation). A yeast-based expression system combines the ease ofgenetic manipulation and fermentation of a microbial organism with thecapability to secrete and to modify proteins. However, recombinantglycoproteins produced in yeast cells exhibit mainly heterogeneoushigh-mannose and hyper-mannose glycan structures, which can bedetrimental to protein function, downstream processing, and subsequenttherapeutic use, particularly where glycosylation plays a biologicallysignificant role.

SUMMARY

The present invention is based, at least in part, on: (a) the discoverythat single gene deletion (Outer CHain elongation (OCH1) deletion) inYarrowia lypolitica cells resulted in the substantially homogeneousproduction of glycosylated proteins having α-1,2-linked mannose residueson a Man₅GlcNAc₂ (structural formula IV; FIG. 1) backbone; (b) thediscovery that overexpression of an engineered alpha-1,2-mannosidasetargeted to the ER of Yarrowia lipolytica cells (both with AND withoutOCH1 deletion) resulted in the substantially homogenous production ofglycosylated proteins carrying the Man₅GlcNAc₂ N-glycan structure(structural formula IV; FIG. 1); (c) the discovery that inactivating theAsparagine Linked Glycosylation 3 (ALG3) enzyme activity in Yarrowialipolytica cells results in highly increased levels of glucosylatedglycans; and (d) the discovery that overexpression of a wild-type formof a Yarrowia lipolytica gene (MNN4) in Yarrowia lipolytica results inhyperphosphorylation of α-1,2-linked mannose residues. Thus, thegenetically engineered cells (e.g., Yarrowia lipolytica, Arxulaadeninivorans, or other related species dimorphic yeast cells) can beused in methods to produce target molecules having an alteredN-glycosylation form as compared to the N-glycosylation form of thetarget molecules produced in non-genetically engineered cells of thesame species. As administration of N-glycosylated target molecules(e.g., N-glycosylated proteins) to patients having a metabolic disorder(e.g., a lysosomal storage disorder) has been shown to ameliorate thesymptoms of the disorder, the methods and cells described are useful forthe preparation of N-glycosylated target molecules for the treatment of,inter alia, metabolic disorders such as lysosomal storage disorders.

The present invention is also based, at least in part, on the discoveryof the spliced form of the Yarrowia lipolytica and Pichia pastoris HAC1gene. The protein encoded by the HAC1 gene, Hac1p, is a transcriptionalactivator that activates transcription of several target genes bybinding to a DNA sequence motif termed the Unfolded Protein Response(UPR) element. Among the Hac1p target genes are those that encodechaperones, foldases, and proteins which are responsible for lipid- andinositol metabolism. As the spliced form Hac1p is a more potenttranscriptional activator than the form encoded by the unspliced HAC1mRNA, overexpression of the spliced form of Hac1p transcription factorcan lead to an increased expression of native and heterologeous proteinsas well as an increase in ER membrane. Thus, the spliced form of Hac1pcan be used to increase the production of membrane and secreted proteinsin a variety of eukaryotic cells (e.g., fungal cells (e.g., Yarrowialipolytica or any other yeast cells described herein), plant cells, oranimal cells (e.g., mammalian cells such as human cells) by simultaneousactivation of the UPR and expression of target molecules.

The present invention is further based on the discovery of a mutant formof the MNS1 mannosidase capable of converting Man₈GlcNAc₂ (structuralformula I; FIG. 4) structures to Man₅GlcNAc₂ (structural formula IV;FIG. 4), Man₆GlcNAc₂ (structural formula V; FIG. 4) and Man₇GlcNAc₂(structural formula VI; FIG. 4) when expressed in Yarrowia lipolytica.Thus, genetically engineered eukaryotic cells (e.g., fungal cells (e.g.,Yarrowia lipolytica or any other yeast cells described herein), plantcells, or animal cells (e.g., mammalian cells such as human cells))expressing mutant forms of mannosidase such as MNS1 can be used inmethods to produce target molecules having an altered N-glycosylationform as compared to the N-glycosylation form of the target moleculesproduced in non-genetically engineered cells of the same species.Therefore, the cells and methods described are useful for thepreparation of N-glycosylated target molecules for the treatment of,inter alia, metabolic disorders such as lysosomal storage disorders (seebelow).

In one aspect, the disclosure features a method of producing an alteredN-glycosylation form of a target protein. The method includes the stepof introducing into a cell a nucleic acid encoding a target protein,wherein the cell produces the target protein in an alteredN-glycosylation form and wherein the cell is a Yarrowia lipolytica or anArxula adeninivorans cell (or a related species dimorphic yeast cell)genetically engineered to contain at least one modified N-glycosylationactivity. The method can also include the step of providing the Yarrowialipolytica or an Arxula adeninivorans cell (or related species dimorphicyeast cell) genetically engineered to contain at least one modifiedN-glycosylation activity. The method can also include the step ofisolating the altered N-glycosylation form of the target protein.

In some embodiments, the target protein can be an endogenous protein oran exogenous protein. The target protein can be a mammalian protein suchas a human protein. The target protein can be, for example, a pathogenprotein, a lysosomal protein, a growth factor, a cytokine, a chemokine,an antibody or antigen-binding fragment thereof, or a fusion protein.The fusion protein can be, for example, a fusion of a pathogen protein,a lysosomal protein, a growth factor, a cytokine, or a chemokine with anantibody or an antigen-binding fragment thereof. The target protein canbe, for example, one associated with a lysosomal storage disorder (LSD).The target protein can be, for example, glucocerebrosidase,galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase,beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase,alpha-L-fucosidase, arylsulfatase B, arylsulfatase A,alpha-N-acteylgalactosaminidase, aspartylglucosaminidase,iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase,beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase,alpha-neuraminidase, phosphotransferase, acid lipase, acid ceramidase,sphinogmyelinase, thioesterase, cathepsin K, or lipoprotein lipase.

In some embodiments, the altered N-glycosylation form can contain one ormore N-glycan structures such as, e.g., Man₅GlcNAc₂, Man₈GlcNAc₂,Man₉GlcNAc₂, Man₃GlcNAc₂, Glc₁Man₅GlcNAc₂, Glc₂Man₅GlcNAc₂. In someembodiments, the altered glycosylation can be, for example, Man₅GlcNAc₂,Man₈GlcNAc₂, Man₉GlcNAc₂, Man₃GlcNAc₂, Glc₁Man₅GlcNAc₂, Glc₂Man₅GlcNAc₂.

In some embodiments, the altered N-glycosylation form of the targetprotein can be homogenous or substantially homogenous. For example, thefraction of altered target molecules that contain the alteredglycosylation can be at least about 20%, at least about 30%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95% or more.

In some embodiments, the cell can be genetically engineered to bedeficient in at least one N-glycosylation activity. The N-glycosylationactivity can be, for example, ALG3 activity, OCH1 activity, MNS1activity, or MNN9 activity.

In some embodiments, at least one modification can be: (a) deletion of agene encoding a protein having the N-glycosylation activity; (b)expression of a mutant form of a protein having the N-glycosylationactivity; (c) introduction or expression of an RNA molecule thatinterferes with the functional expression of a protein having theN-glycosylation activity; (d) expression of a protein havingN-glycosylation activity (such as ALG6 or an alpha-mannosidase (e.g., analpha-mannosidase targeted to the endoplasmic reticulum). The expressedprotein can be a protein encoded by an exogenous nucleic acid in thecell. The expressed protein can be an alpha-mannosidase with a pHoptimum below 7.5 (e.g., a pH optimum below 5.1). The protein havingN-glycosylation activity can be an exogenous protein. The protein havingN-glycosylation activity can be a mammalian protein (such as a humanprotein) or a lower eukaryotic (e.g., a fungus, a protozoan, or atrypanosome) protein. The lower eukaryote can be selected from the groupconsisting of Typanosoma brucei, Trichoderma harzianum, an Aspergillus,and any other lower eukaryote described herein.

In some embodiments, the N-glycosylation activity can be aglucosyltransferase activity. In some embodiments, the protein havingN-glycosylation activity is ALG6 or an alpha-mannosidase. Thealpha-mannosidase can be targeted to the endoplasmic reticulum. Forexample, the protein having N-glycosylation activity can be a fusionprotein comprising an alpha-mannosidase polypeptide and an HDELendoplasmic reticulum retention peptide.

In some embodiments, the protein having N-glycosylation activity can bea protein that is capable of removing glucose residues from Man₅GlcNAc₂.For example, the protein having N-glycosylation activity can be aprotein having α-1,3-glucosidase activity such as, but not limited to, aglucosidase II (e.g., one or both of the alpha and beta subunit of aglucosidase II) or a mutanase.

In some embodiments, the cell can be genetically engineered to compriseat least two modified N-glycosylation activities such as any of themodified N-glycosylation activities described herein. The at least twomodified N-glycosylation activities can comprise, e.g., a deficiency inan ALG3 activity and an elevated level of an ALG6 activity.

In some embodiments, the cell can be genetically engineered to compriseat least three modified N-glycosylation activities such as any of themodified N-glycosylation activities described herein. The at least threemodified N-glycosylation activities can comprise, e.g., a deficiency inan ALG3 activity; an elevated level of an ALG6 activity; and an elevatedlevel of a a glucosidase II activity.

In some embodiments, the cell is not genetically engineered to bedeficient in an OCH1 activity.

In some embodiments, modification can comprise expression of a proteinor biologically active variant thereof capable of effecting mannosylphosphorylation of the target protein. The protein or biologicallyactive variant thereof capable of effecting mannosyl phosphorylation canbe MNN4, PNO1, or MNN6. In some embodiments, at least about 30% of themannosyl residues of a glycoprotein can be phosphorylated.

In some embodiments, the method can further include additionalprocessing of the glycoprotein. The additional processing can occur invitro or in vivo. The additional processing can comprise addition of aheterologous moiety to the modified glycoprotein. The heterologousmoiety can be a polymer or a carrier. The additional processing cancomprise enzymatic or chemical treatment of the altered N-glycosylationform of the target protein. For example, the additional processing cancomprise treatment of the altered N-glycosylation form of the targetprotein with a mannosidase, a mannanase, a phosphodiesterase, aglucosidase, or a glycosyltransferase. The additional processing caninclude treatment of the altered N-glycosylation form of the targetprotein with hydrofluoric acid. The additional processing can includephosphorylation of the altered N-glycosylation form of the targetprotein.

In another aspect, the disclosure provides a method of producing analtered N-glycosylation form of a target protein. The method includesthe steps of: providing a eukaryotic cell (e.g., a fungal cell, a plantcell, or an animal cell) genetically engineered to comprise at least onemodified N-glycosylation activity; and introducing into the cell anucleic acid encoding a target protein, wherein the cell produces thetarget protein in an altered N-glycosylation form.

In another aspect, the disclosure features a method of producing analtered N-glycosylation form of a target protein. The method includesthe step of contacting a target protein with a cell lysate prepared froma Yarrowia lipolytica or an Arxula adeninivorans cell geneticallyengineered to comprise at least one modified N-glycosylation activity,wherein the contacting of the target protein with the cell lysateresults in an altered N-glycosylation form of the target protein.

In yet another aspect, the disclosure features a method of producing analtered N-glycosylation form of a target protein, which method includesthe step of contacting a target protein with one or more proteins havingN-glycosylation activity, wherein the one or more proteins havingN-glycosylation activity are obtained from a Yarrowia lipolytica or anArxula adeninivorans cell genetically engineered to comprise at leastone modified N-glycosylation activity and wherein contacting the targetmolecule with the one or more proteins having N-glycosylation activityresults in an altered N-glycosylation form of the target protein.

In another aspect, the disclosure provides an isolated protein havingaltered N-glycosylation, wherein the protein is produced by any of themethods described above.

In yet another aspect, the disclosure provides an isolated Yarrowialipolytica or Arxula adeninivorans cell (or other related speciesdimorphic yeast cell) genetically engineered to comprise at least onemodified N-glycosylation activity. The N-glycosylation activity can be,for example, ALG3 activity, OCH1 activity, MNS1 activity, or MNN9activity. The modification can be any of those described herein. Forexample, the modification can include: (a) deletion of a gene encoding aprotein having the N-glycosylation activity, (b) expression of a mutantform of a protein having the N-glycosylation activity, (c) introductionor expression of an RNA molecule that interferes with the functionalexpression of a protein having the N-glycosylation activity, or (d)expression of a protein having N-glycosylation activity. The proteinhaving N-glycosylation activity can be, for example, ALG6. The proteinhaving N-glycosylation activity can be a mammalian protein such as ahuman protein. The modification can also include expression of a protein(e.g., MNN4 or PNO1) or biologically active variant thereof capable ofpromoting mannosyl phosphorylation of the modified glycoprotein.

In another aspect, the disclosure provides a method of treating adisorder treatable by administration of a protein having alteredN-glycosylation. The method includes the steps of administering to asubject a protein obtained by any of the methods described above,wherein the subject is one having, or suspected of having, a diseasetreatable by administration of a protein having altered N-glycosylation.The method can also include the steps of (a) providing a subject and/or(b) determining whether the subject has a disease treatable byadministration of a protein having altered N-glycosylation. The subjectcan be mammal such as a human. The disorder can be, for example, acancer, an immunological disorder (e.g., an inflammatory condition) or ametabolic disorder. The metabolic disorder can be any of those describedherein, e.g., a lysosomal storage disorder (LSD) such as Gaucherdisease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, orFabry disease. The protein can be one associated with an LSD, e.g., theprotein can be, for example, glucocerebrosidase, alpha-galactosidase.The protein can be, for example, alpha-L-iduronidase,beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase,beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfataseA, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase,iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase,beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase,alpha-neuromimidase, phosphotransferase, acid lipase, acid ceramidase,sphinogmyelinase, thioesterase, cathepsin K, or lipoprotein lipase.

In another aspect, the disclosure provides a substantially pure cultureof Yarrowia lipolytica or Arxula adeninivorans cells (or other relatedspecies dimorphic yeast cells), a substantial number of which beinggenetically engineered to comprise at least one modified N-glycosylationactivity (such as any of the modifications described herein). Theculture of cells can contain one or more subpopulations of cells, eachsubpopulation comprising a different modified glycosylation activity.

In yet another aspect, the disclosure provides: (a) an isolatednucleotide sequence comprising SEQ ID NO:1 or SEQ ID NO:2; (b) anisolated nucleotide sequence comprising a sequence that is at least 80%identical to SEQ ID NO:1 or SEQ ID NO:2; or (c) a polypeptide encoded bythe isolated nucleotide sequence of (a) or (b). In some embodiments, theisolated nucleic acid sequence is SEQ ID NO:1 or SEQ ID NO:2.

In another aspect, the disclosure features an isolated nucleic acidcontaining: (a) a nucleotide sequence that hybridizes under highlystringent conditions to the complement of SEQ ID NO:1 or SEQ ID NO:2; or(b) the complement of the nucleotide sequence.

In yet another aspect, the disclosure provides: (a) an isolatednucleotide sequence comprising (or consisting of) any of the nucleicacid sequences depicted herein; (b) an isolated nucleotide sequencecomprising a sequence that is at least 80% identical to any of thenucleic acid sequences depicted herein; or (c) a polypeptide encoded bythe isolated nucleotide sequence of (a) or (b). In some embodiments, theisolated nucleic acid sequence is any of the nucleic acid sequencesdepicted herein.

In another aspect, the disclosure features an isolated nucleic acidcontaining: (a) a nucleotide sequence that hybridizes under highlystringent conditions to the complement of any of the nucleic acidsequences depicted herein; or (b) the complement of the nucleotidesequence.

In yet another aspect, the disclosure provides: (a) a vector comprisingany of the nucleic acid sequences described above or (b) a cultured cellcontaining the vector of (a). The vector can be an expression vector.The nucleic acid sequence in the vector can be operably linked toexpression control sequence.

In another aspect, the disclosure provides a method for producing aprotein. The method includes the step of culturing any of the cellsdescribed above under conditions permitting the expression of thepolypeptide. The method can also include the step of, after culturingthe cell, isolating the polypeptide from the cell or the medium in whichthe cell was cultured. The cell can be, e.g., a cultured cell containinga vector comprising any of the nucleic acid sequences described above.

The target molecules (e.g., target proteins), proteins havingN-glycosylation activity, and altered N-glycosylation moleculesdescribed herein (collectively referred to as “molecules of theinvention”) can, but need not, be isolated. The term “isolated” asapplied to any of the molecules of the invention described herein refersto a molecule, or a fragment thereof, that has been separated orpurified from components (e.g., proteins or other naturally-occurringbiological or organic molecules) which naturally accompany it. It isunderstood that recombinant molecules (e.g., recombinant proteins) willalways be “isolated.” Typically, a molecule of the invention is isolatedwhen it constitutes at least 60%, by weight, of the total molecules ofthe same type in a preparation, e.g., 60% of the total molecules of thesame type in a sample. For example, an altered glycosylation protein isisolated when it constitutes at least 60%, by weight, of the totalprotein in a preparation or sample. In some embodiments, a molecule ofthe invention in the preparation consists of at least 75%, at least 90%,or at least 99%, by weight, of the total molecules of the same type in apreparation.

As used herein, an “altered N-glycosylation form” of a target moleculeis an N-glycosylation form of a target molecule produced by agenetically engineered host cell (e.g., Yarrowia lipolytica cell, Arxulaadeninivorans cell, or a cell of another related dimorphic yeast cellspecies) that differs from the N-glycosylation form of the targetmolecule produced in a non-genetically engineered cell of the samespecies as the genetically engineered cell. Thus, an alteredglycosylation form of a target molecule can be, for example, a form ofthe target molecule that is not N-glycosylated. Moreover, an alteredglycosylation form of a target molecule can be, e.g., a form of thetarget molecule that has altered phosphorylation of one or more N-linkedglycans.

As used herein, the term “other related dimorphic yeast cell species”refers to yeasts related to Yarrowia lipolytica and Arxula adeninivoransthat belong to the family Dipodascaceae such as Arxula, Dipodascus (e.g.D. albidus, D. ingens, or D. specifer), Galactomyces (e.g. G. reesii orG. geotrichum), Sporopachyderma, Stephanoascus (e.g., S. ciferii),Wickerhamiella, and Zygoascus. Specifically, yeasts in the GladeMetchnikowia (e.g., M. pulcherrima or M. agaves) and Stephanoascus (towhich Y. lipolytica is assigned by analysis of the D1/D2 domain of the26S-rDNA sequences of species such as Arxula (e.g. A. adeninivorans orA. terrestris)) and some Candida species (e.g., C. apicola but not C.albicans, C. maltosa, or C. tropicalis).

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification.

The disclosure also provides (i) biologically active variants and (ii)biologically active fragments or biologically active variants thereof,of the wild-type, full-length, mature “target proteins” or “proteinshaving N-glycosylation activity” described herein. Biologically activevariants of full-length, mature, wild-type proteins or fragments of theproteins can contain additions, deletions, or substitutions. Proteinswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Aconservative substitution is the substitution of one amino acid foranother with similar characteristics. Conservative substitutions includesubstitutions within the following groups: valine, alanine and glycine;leucine, valine, and isoleucine; aspartic acid and glutamic acid;asparagine and glutamine; serine, cysteine, and threonine; lysine andarginine; and phenylalanine and tyrosine. The non-polar hydrophobicamino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Any substitution of one memberof the above-mentioned polar, basic or acidic groups by another memberof the same group can be deemed a conservative substitution. Bycontrast, a non-conservative substitution is a substitution of one aminoacid for another with dissimilar characteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids.

Additions (addition variants) include fusion proteins containing: (a)full-length, wild-type, mature polypeptides or fragments thereofcontaining at least five amino acids; and (b) internal or terminal (C orN) irrelevant or heterologous amino acid sequences. In the context ofsuch fusion proteins, the term “heterologous amino acid sequences”refers to an amino acid sequence other than (a). A fusion proteincontaining a peptide described herein and a heterologous amino acidsequence thus does not correspond in sequence to all or part of anaturally occurring protein. A heterologous sequence can be, for examplea sequence used for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagluttanin (HA),glutathione-S-transferase (GST), or maltose-binding protein (MBP)).Heterologous sequences can also be proteins useful as diagnostic ordetectable markers, for example, luciferase, green fluorescent protein(GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments,the fusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response (e.g.,for antibody generation; see below) or endoplasmic reticulum or Golgiapparatus retention signals. Heterologous sequences can be of varyinglength and in some cases can be a longer sequences than the full-lengthtarget proteins to which the heterologous sequences are attached.

A “fragment” as used herein, refers to a segment of the polypeptide thatis shorter than a full-length, immature protein. Fragments of a proteincan have terminal (carboxy or amino-terminal) and/or internal deletions.Generally, fragments of a protein will be at least four (e.g., at leastfive, at least six, at least seven, at least eight, at least nine, atleast 10, at least 12, at least 15, at least 18, at least 25, at least30, at least 35, at least 40, at least 50, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, or atleast 100 or more) amino acids in length.

Biologically active fragments or biologically active variants of thetarget proteins or proteins having N-glycosylation activity have atleast 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%;95%; 97%; 98%; 99%; 99.5%, or 100% or even greater) of the activity ofthe wild-type, full-length, mature protein. In the case of a targetprotein, the relevant activity is the ability of the target protein toundergo altered N-glycosylation in a genetically engineered cell. In thecase of a protein having N-glycosylation activity, the relevant activityis N-glycosylation activity.

Depending on their intended use, the proteins, biologically activefragments, or biologically active variants thereof can be of anyspecies, such as, e.g., fungus (including yeast), nematode, insect,plant, bird, reptile, or mammal (e.g., a mouse, rat, rabbit, hamster,gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, or human). Insome embodiments, biologically active fragments or biologically activevariants include immunogenic and antigenic fragments of the proteins. Animmunogenic fragment is one that has at least 25% (e.g., at least: 30%;40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%, or100% or even more) of the ability of the relevant full-length, immatureprotein to stimulate an immune response (e.g., an antibody response or acellular immune response) in an animal of interest. An antigenicfragment of a protein is one having at least 25% (e.g., at least: 30%;40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%, or100% or even greater) of the ability of the relevant full-length,immature protein to be recognized by an antibody specific for theprotein or a T cell specific to the protein.

“N-glycosylation activity” as used herein refers to any activity that is(i) capable of adding N-linked glycans to a target molecule (i.e., anoligosaccharyltransferase activity); (ii) removing N-linked glycans froma target molecule, (iii) modifying one or more N-linked glycans on atarget molecule, (iv) modifying dolichol-linked oligosaccharides; or (v)is capable of aiding the activity of the activities under (i-iv). Assuch, N-glycosylation activity includes, e.g., N-glycosidase activity,glycosidase activity, glycosyltransferase activity, sugar nucleotidesynthesis, modification, or transporter activity. Modification of one ormore N-linked glycans on a target molecule includes the action of amannosylphosphoryltransferase activity, a kinase activity, or aphosphatase activity, e.g., a mannosylphosphoryltransferase, a kinase,or a phosphatase activity that alters the phosphorylation state ofN-linked glycans on target molecules.

As used herein, to “genetically engineer” a cell or a “geneticallyengineered cell” and like terminology refers to any artificially createdgenetic alteration of a cell that results in at least one modifiedN-glycosylation activity in the cell as compared to a non-geneticallyengineered cell (e.g., a fungal cell such as Yarrowia lipolytica cell,Arxula adeninivorans cell, or other related species dimorphic yeastcell, a plant cell, or an animal cell (e.g., a mammalian cell such as ahuman cell)). Thus, it is understood that artificially created geneticalterations do not include, e.g., spontaneous mutations. Examples ofartificial genetic alterations are described below (see “GeneticallyEngineered Cells”).

As used herein, the term “wild-type” as applied to a nucleic acid orpolypeptide refers to a nucleic acid or a polypeptide that occurs in, oris produced by, respectively, a biological organism as that biologicalorganism exists in nature.

The term “heterologous” as applied herein to a nucleic acid in a hostcell or a polypeptide produced by a host cell refers to any nucleic acidor polypeptide (e.g., an protein having N-glycosylation activity) thatis not derived from a cell of the same species as the host cell.Accordingly, as used herein, “homologous” nucleic acids, or proteins,are those that occur in, or are produced by, a cell of the same speciesas the host cell.

The term “exogenous” as used herein with reference to nucleic acid and aparticular host cell refers to any nucleic acid that does not occur in(and cannot be obtained from) that particular cell as found in nature.Thus, a non-naturally-occurring nucleic acid is considered to beexogenous to a host cell once introduced into the host cell. It isimportant to note that non-naturally-occurring nucleic acids can containnucleic acid subsequences or fragments of nucleic acid sequences thatare found in nature provided that the nucleic acid as a whole does notexist in nature. For example, a nucleic acid molecule containing agenomic DNA sequence within an expression vector isnon-naturally-occurring nucleic acid, and thus is exogenous to a hostcell once introduced into the host cell, since that nucleic acidmolecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of yeast x is an exogenousnucleic acid with respect to a cell of yeast y once that chromosome isintroduced into a cell of yeast y.

It will be clear from the above that “exogenous” nucleic acids can be“homologous” or “heterologous” nucleic acids. In contrast, the term“endogenous” as used herein with reference to nucleic acids or genes (orproteins encoded by the nucleic acids or genes) and a particular cellrefers to any nucleic acid or gene that does occur in (and can beobtained from) that particular cell as found in nature.

As an illustration of the above concepts, an expression plasmid encodinga Y. lipolytica ALG6 protein that is transformed into a Y. lipolyticacell is, with respect to that cell, an exogenous nucleic acid. However,the ALG6 protein coding sequence and the ALG6 protein produced by it arehomologous with respect to the cell. Similarly, an expression plasmidencoding a Arxula adeninivorans ALG6 protein that is transformed into aY. lipolytica cell is, with respect to that cell, an exogenous nucleicacid. In contrast with the previous example, however, the ALG6 proteincoding sequence and the ALG6 protein produced by it are heterologouswith respect to the cell.

As used herein, a “promoter” refers to a DNA sequence that enables agene to be transcribed. The promoter is recognized by RNA polymerase,which then initiates transcription. Thus, a promoter contains a DNAsequence that is either bound directly by, or is involved in therecruitment, of RNA polymerase. A promoter sequence can also include“enhancer regions,” which are one or more regions of DNA that can bebound with proteins (namely, the trans-acting factors, much like a setof transcription factors) to enhance transcription levels of genes(hence the name) in a gene-cluster. The enhancer, while typically at the5′ end of a coding region, can also be separate from a promoter sequenceand can be, e.g., within an intronic region of a gene or 3′ to thecoding region of the gene.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

Variants of any of the nucleic acid sequences described herein (e.g.,the HAC1 sequences as depicted in SEQ ID NO:1 or SEQ ID NO:2) can have asequence that is homologous, e.g., a sequence bearing at least about 70%(e.g., at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 99%) homologous(identical) to the wild-type nucleic acid sequence. Such wild-typesequences can be isolated from nature or can be produced by recombinantor synthetic methods. Thus a wild-type sequence nucleic acid can havethe nucleic acid sequence of naturally occurring human nucleic acidsequences, monkey nucleic acid sequences, murine nucleic acid sequences,or any other species that contains a homologue of the wild-type nucleicacid of interest. As used herein, a “homologous” or “homologous nucleicacid sequence” or similar term, refers to sequences characterized byhomology at the nucleotide level of at least a specified percentage andis used interchangeably with sequence identity.

Percent homology or identity can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for UNIX,Genetics Computer Group, University Research Park, Madison, Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman ((1981)Adv. Appl. Math. 2:482-489). In some embodiments, homology between aprobe and target (see below) is between about 50% to about 60%. In someembodiments, homology between a probe and target nucleic acid is betweenabout 55% to 65%, between about 65% to 75%, between about 70% to 80%,between about 75% and 85%, between about 80% and 90%, between about 85%and 95%, or between about 90% and 100%.

The term “probe,” as used herein, refers to nucleic acid sequences ofvariable length. In some embodiments, probes comprise at least 10 and asmany as 6,000 nucleotides. In some embodiments probes comprise at least12, at lease 14, at least 16, at least 18, at least 20, at least 25, atleast 50 or at least 75 or 100 contiguous nucleotides. Longer lengthprobes are usually obtained from natural or recombinant sources (asopposed to direct, chemical synthesis), are highly specific to thetarget sequence, and are much slower to hybridize to the target thanlonger oligomers. Probes can be single or double stranded nucleic acidmolecules.

In some embodiments, a variant nucleic acid described herein can have asequence comprising one or both strands with partial complementary(e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% complementary) to a region, portion, domain, or segment of thewild-type nucleic acid of interest (e.g., the HAC1 nucleic acidsequences as depicted in SEQ ID NO:1 or SEQ ID NO:2). In someembodiments, a variant nucleic acid sequence of interest can have asequence comprising one or both strands with full complementary (i.e.,100% complementary) to a region, portion, domain, or segment of thewild-type nucleic acid sequence. Sequence “complementarity” refers tothe chemical affinity between specific nitrogenous bases as a result oftheir hydrogen bonding properties (i.e., the property of two nucleicacid chains having base sequences such that an antiparallel duplex canform where the adenines and uracils (or thymine, in the case of DNA ormodified RNA) are apposed to each other, and the guanines and cytosinesare apposed to each other). Fully complementary sequences, thus, wouldbe two sequences that have complete one-to-one correspondence (i.e.,adenine to uracil/thymidine and guanine to cytosine) of the basesequences when the nucleotide sequences form an antiparallel duplex.

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A nucleic acid sequence described herein, or afragment or variant thereof, can be used as a hybridization probeaccording to standard hybridization techniques. The hybridization of acertain probe of interest (e.g., a probe of a HAC1 nucleotide sequence,e.g., the HAC1 nucleotide sequences as depicted in SEQ ID NOS:1 or 2) toDNA or RNA from a test source (e.g., a eukaryotic cell) is an indicationof the presence of DNA or RNA (e.g., a HAC1 nucleotide sequence)corresponding to the probe in the test source. Hybridization conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6,1991. Moderate hybridization conditions are defined as equivalent tohybridization in 2× sodium chloride/sodium citrate (SSC) at 30° C.,followed by a wash in 1×SSC, 0.1% SDS at 50° C. Highly stringentconditions are defined as equivalent to hybridization in 6× sodiumchloride/sodium citrate (SSC) at 45° C., followed by a wash in 0.2×SSC,0.1% SDS at 65° C.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, Genbank® Accession Nos, and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present application, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention, e.g., methods ofproducing altered N-glycosylation molecules, will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting N-glycan precursor synthesis atthe yeast endoplasmic reticulum. Genes whose encoded protein has anactivity mediating the indicated enzymatic conversions are in shadedboxes (e.g., ALG7; upper left). “UDP” and “UMP” refer to uridinediphosphate and uridine monophosphate, respectively. “GDP” and “GMP”refer to guanosine diphosphate and guanosine monophosphate respectively.“Gn” refers to N-acetylglucosamine. “M” refers to monomeric mannose, Grefers to glucose, Pi refers to phosphate.

FIG. 2 is a schematic diagram depicting N-glycan processing in the yeastendoplasmic reticulum.

FIG. 3 is a schematic diagram depicting N-glycan processing in the S.cerevisiae Golgi apparatus. Genes whose encoded protein has an activitymediating the indicated enzymatic conversions are in shaded boxes (e.g.,OCH1; middle left).

FIGS. 4A and 4B are a schematic diagrams depicting the structure of thevarious N-glycan structures described herein.

FIGS. 5A-5E are schematic diagrams depicting the cloning strategy forOCH1 gene disruption in Yarrowia lipolytica. “PCR” refers to polymerasechain reaction.

FIGS. 6A and 6B are schematic diagrams depicting the cloning strategyfor MNN9 gene disruption fragment. “PCR” refers to polymerase chainreaction.

FIG. 7 is a series of electroferograms depicting N-glycan analysis ofmannoproteins obtained from wild-type Yarrowia lipolytica cells orglycosylation mutant (e.g., Δoch1 cI9, Δmnn9 1 and Δoch1 Δmnn9) cellsand MTLY60 strain cells. In some cases, the N-glycans were furthertreated with α-1,2 mannosidase. Analysis was performed using DNAsequencer-assisted, fluorophore-assisted carbohydrate electrophoresis(DSA-FACE). “M5,” “M6,” “M7,” “M8,” and “M9,” refer to the number ofmannose residues conjugated to the base N-acetylglucosamine structure.The Y-axis represents the relative fluorescence units as an indicationof the amount of each of the mannose structures. The X-axis representsthe relative mobility of each complex mannose structure through a gel.The top electroferogram is an analysis of oligomaltose for use as amobility standard.

FIGS. 8A-8E are schematic diagrams depicting the cloning strategy for S.cerevisiae MNS1 expression vector. “PCR” refers to polymerase chainreaction.

FIG. 9 is a series of electroferograms depicting N-glycan analysis ofsecreted glycoproteins obtained from MTLY60 cells expressing wild-type(WT) Mns1p or various mutant forms (i.e., R273G, R273L, orR269S/S272G/R273L) of Mns1p as indicated. Analysis was performed usingDSA-FACE. “M5,” “M6,” “M7,” “M8,” “M9,” refers to the number of mannoseresidues conjugated to the base N-acetylglucosamine structure. TheY-axis represents the relative fluorescence units as an indication ofthe amount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through a gel. Thetop electroferogram is an analysis of oligomaltose for use as a mobilitystandard.

FIG. 10 is a schematic diagram depicting the cloning strategy for anMNN4 expression vector.

FIG. 11 is a series of electroferograms depicting N-glycan analysis ofsecreted glycoproteins obtained from wild-type MTLY60 cells orglycosylation mutant cells as indicated. Analysis was performed usingDSA-FACE. “M5,” “M6,” “M7,” “M8,” “M9,” refers to the number of mannoseresidues conjugated to the chitobiose core structure. “P” refers tomannoproteins containing one phosphate residue and “PP” refers tomannoproteins containing two phosphate residues. The Y-axis representsthe relative fluorescence units as an indication of the amount of eachof the mannose structures. The X-axis represents the relative mobilityof each complex mannose structure through a gel. The top electroferogramis an analysis of oligomaltose for use as a mobility standard.

FIGS. 12A-12H are schematic diagrams depicting the cloning strategy foran α-galactosidase expression vector. The nucleotide sequences set forthin SEQ ID NOs: 76-81 are the oligonucleotides used in the cloningstrategy in FIGS. 12A, 12C, and 12E.

FIG. 13 is a series of electroferograms depicting N-glycan analysis ofmannoproteins and phosphomannoproteins obtained from wild-type MTLY60cells or various clones of glycosylation mutant cells as indicated.“alg3” indicates that the cell is an ALG3 knockout. “ALG6overexpression” indicates that the protein product of ALG6 isoverexpressed in the cell. Analysis was performed using DSA-FACE. “M5,”“M6,” “M7,” “M8,” and “M9,” refer to the number of mannose residuesconjugated to the base N-acetylglucosamine structure. The Y-axisrepresents the relative fluorescence units as an indication of theamount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through apolyacrylamide gel. The top electroferogram is an analysis ofoligomaltose for use as a mobility standard.

FIG. 14 is a series of electroferograms depicting N-glycan analysis ofmannoproteins and phosphomannoproteins obtained from wild-type MTLY60cells or various clones of glycosylation mutant cells as indicated.“alg3” indicates that the cell is an ALG3 knockout. “ALG6overexpression” indicates that the protein product of ALG6 isoverexpressed in the cell. One peak runs at the same position asMan₅GlcNAc₂ of the RNaseB marker and shifts with two glucose-units afterα-1,2-mannosidase treatment and with 4 glucose-units afteralpha-mannosidase (JB) digest. This fits with a Man₅GlcNAc₂ structure asexpected. The additional two peaks run at a distance of about one andtwo glyco-units and are not affected by a-1,2-mannosidase digestion.Both peaks shift one glucose-unit upon a-mannosidase (JB) digestion.Minor shifts are due to the higher salt concentrations of the addedenzymes, e.g. JB mannosidase. Analysis was performed using DSA-FACE.“M5,” “M6,” “M7,” “M8,” and “M9,” refer to the number of mannoseresidues conjugated to the chitobiose core structure. The Y-axisrepresents the relative fluorescence units as an indication of theamount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through a gel. Thetop electroferogram is an analysis of oligomaltose for use as a mobilitystandard.

FIG. 15 is a sequence alignment of an isolated DNA fragment (SEQ IDNO:1) sequence obtained from the unfolded protein response (UPR)-inducedstrain Yarrowia lipolytica with a genomic HAC1 DNA sequence (SEQ IDNO:5). The boxed sequence corresponds to the non-conventionally splicedintron.

FIG. 16 is a series of sequence alignments of the predicted 5′ (top) and3′ (bottom) splice sites of Pichia pastoris (SEQ ID NO: 70 and SEQ IDNO: 72) and Saccharomyces cerevisiae (SEQ ID NO: 71 and SEQ ID NO: 73).Nucleotides in bold underlined are present in the loop structure.

FIGS. 17A and 17B are two partial views of a sequence alignment of theHAC1 cDNA obtained from DTT-induced (I) (SEQ ID NO:2) and non-induced(NI) (SEQ ID NO:6) Pichia pastoris cultures.

FIG. 18 is a sequence alignment of the 18 amino acid C-terminal regionsof Pichia pastoris (SEQ ID NO: 74) and Saccharomyces cerevisiae (SEQ IDNO: 75). Conserved amino acids are in bold and underlined.

FIG. 19 is a bar graph depicting the comparison of the relativeexpression levels of KAR2 mRNA. Clones 3, 4, and 5 (Pichia pastoris GSM5cells) were grown on methanol as carbon source. “3+,” “4+,” and “5+”refer to the respective clones grown on methanol as carbon source,whereas “3−,” “4−,” and “5−” refer to the respective clones grown onglucose as carbon source. The Y-axis represents the relative expressionof the KAR2 gene using real-time PCR.

FIG. 20 is a bar graph depicting the relative expression level of Kar2and HAC1 mRNA in two Pichia pastoris clones (clone 6 and 8). “6+” and“8+” refer to the respective clones grown on methanol as carbon source,whereas “6−” and “8−” refer to the respective clones grown on glucose ascarbon source. The Y-axis represents the relative expression of the KAR2gene using real-time PCR.

FIG. 21 is a schematic diagram depicting the cloning strategy for aYlMNN6 expression vector.

FIG. 22 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from Δoch1 Y. lipolytica cells, alone, or variousclones (Z3, Z4, Z5, U5, U6, and U8) of Δoch1 Y. lipolytica expressingYlMNN6 as indicated. Analysis was performed using DSA-FACE. The Y-axisrepresents the relative fluorescence units as an indication of theamount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through a gel. Thetop electroferogram is an analysis of oligomaltose for use as a mobilitystandard.

FIG. 23 is a schematic diagram depicting the cloning strategy for anMFManHDEL expression vector.

FIG. 24 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from Δoch1 Y. lipolytica cells, alone, or variousclones (9, 11, 10, 3, 5, and 6) of Δoch1 Y. lipolytica expressingMFManHDEL as indicated. Analysis was performed using DSA-FACE. TheY-axis represents the relative fluorescence units as an indication ofthe amount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through a gel. Thetop electroferogram is an analysis of oligomaltose for use as a mobilitystandard.

FIGS. 25A and 25B are schematic diagrams depicting the cloning strategyfor an LIP2preManHDEL expression vector.

FIG. 26 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from Δoch1 Y. lipolytica cells, alone, or variousclones (1, 5, 10, and 11) of Δoch1 Y. lipolytica expressing LIP2ManHDELas indicated. Analysis was performed using DSA-FACE. “M5,” “M6,” “M7,”“M8,” and “M9,” refer to the number of mannose residues conjugated tothe chitobiose core structure. The Y-axis represents the relativefluorescence units as an indication of the amount of each of the mannosestructures. The X-axis represents the relative mobility of each complexmannose structure through a gel. The top electroferogram is an analysisof oligomaltose for use as a mobility standard.

FIGS. 27A and 27B are amino acid sequences of HAC1 proteins of Yarrowialipolytica (FIG. 27A; SEQ ID NO:3) and Pichia pastoris (FIG. 27B; SEQ IDNO:4).

FIG. 28 is a photograph of a Coomassie blue stained polyacrylamide geldepicting the results of Lip2p overexpression in various Yarrowialipolytica cell (MTLY60, MTLY60Δalg3 and MTLY60Δalg3ALG6) cultures. Thefollowing samples were resolved in the gel: Lane 1 (“ladder”), acombination of proteins of known molecular weight; Lane 2 (“WT”), Lip2pprotein obtained from WT Yarrowia lipolytica cells (MTLY60)overexpressing Lip2p; Lane 3 (“WT+PGase F”), Lip2p protein obtained fromWT Yarrowia lipolytica cells overexpressing Lip2p and treated withPNGase F enzyme; Lane 4 (“alg3-ALG6”), Lip2p protein obtained fromYarrowia cells deficient in alg3 and overexpressing both Lip2p and ALG6(MTLY60Δalg3ALG6); Lane 5 (“alg3-ALG6+PNGase F”), Lip2p protein obtainedfrom Yarrowia cells deficient in alg3 and overexpressing both Lip2p andALG6 (MTLY60Δalg3ALG6) and treated with PNGase F enzyme; Lane 6(“alg3”), Lip2p protein obtained from Yarrowia lipolytica cellsdeficient in alg3 and overexpressing Lip2p (MTLY60Δalg3); Lane 7(“alg3+PNGase F”), Lip2p protein obtained from Yarrowia lipolytica cellsdeficient in alg3 and overexpressing Lip2p (MTLY60Δalg3) treated withPNGase F enzyme; Lane 8 (“WT without Lip2p overexpression”), proteinobtained from MTLY60 cells; and Lane 9 (“WT without Lip2poverexpression+PNGase F”), protein obtained from MTLY60 cells andtreated with PNGase F enzyme.

FIG. 29 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (WT(MTLY60); Δalg3; Δalg3 ALG6 overexpressing; and clones of Δalg3overexpressing ALG6 along with the alpha subunit of glucosidase II fromY. lipolytica (Yl) or Typanosoma brucei (Tb)) as indicated. Analysis wasperformed using DSA-FACE. “M5,” “M6,” “M7,” “M8,” and “M9,” refer to thenumber of mannose residues conjugated to the chitobiose core structure.The Y-axis represents the relative fluorescence units as an indicationof the amount of each of the mannose structures. The X-axis representsthe relative mobility of each complex mannose structure through a gel.The top electroferogram is an analysis of oligomaltose for use as amobility standard. The bottom electroferogram is an analysis of RNAse B.

FIG. 30 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3;Δalg3 ALG6 overexpressing; and clones of Δalg3 overexpressing ALG6 alongwith the alpha subunit of glucosidase II from Y. lipolytica (Yl)containing an HDEL sequence as indicated. Analysis was performed usingDSA-FACE. The Y-axis represents the relative fluorescence units as anindication of the amount of each of the mannose structures. The X-axisrepresents the relative mobility of each complex mannose structurethrough a gel.

FIG. 31 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3;Δalg3 ALG6 overexpressing; and clones of Δalg3 overexpressing ALG6 alongwith the alpha subunit of glucosidase II from Trypanosoma brucei (Tb)containing an HDEL sequence) as indicated. Analysis was performed usingDSA-FACE. The Y-axis represents the relative fluorescence units as anindication of the amount of each of the mannose structures. The X-axisrepresents the relative mobility of each complex mannose structurethrough a gel.

FIG. 32 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from alg3ALG6 Yarrowia lipolytica cells treatedin vitro with different concentrations of mutanase as indicated.Analysis was performed using DSA-FACE. The Y-axis represents therelative fluorescence units as an indication of the amount of each ofthe mannose structures. The X-axis represents the relative mobility ofeach complex mannose structure through a gel. The top electroferogram isan analysis of oligomaltose for use as a mobility standard. The bottomelectroferogram is an analysis of RNAse B.

FIG. 33 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3;Δalg3 ALG6 overexpressing; and clones of Δalg3 overexpressing ALG6 alongwith the alpha subunit of glucosidase II from Y. lipolytica (Y.l.) andthe beta subunit of glucosidase II from Y.l. expressed under the controlof Hp4d or TEF promoters) as indicated. The Y-axis represents therelative fluorescence units as an indication of the amount of each ofthe mannose structures. The X-axis represents the relative mobility ofeach complex mannose structure through a gel. The top electroferogram isan analysis of oligomaltose for use as a mobility standard. The bottomelectroferogram is an analysis of RNAse B.

FIG. 34 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3ALG6 overexpressing; and clones of Δalg3 overexpressing ALG6 along withthe HDEL-containing alpha subunit of glucosidase II from Y. lipolytica(Y.l.) and the beta subunit of glucosidase II from Y.l. expressed underthe control of Hp4d or TEF promoters) as indicated. Analysis wasperformed using DSA-FACE. The Y-axis represents the relativefluorescence units as an indication of the amount of each of the mannosestructures. The X-axis represents the relative mobility of each complexmannose structure through a gel. The top electroferogram is an analysisof oligomaltose for use as a mobility standard. The bottomelectroferogram is an analysis of RNAse B.

FIG. 35 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3 andclones of Δalg3 overexpressing the alpha subunit of glucosidase II fromY. lipolytica (Y.l.) and the beta subunit of glucosidase II from Y.l.expressed under the control of a TEF promoter) as indicated. The Y-axisrepresents the relative fluorescence units as an indication of theamount of each of the mannose structures. The X-axis represents therelative mobility of each complex mannose structure through a gel. Thetop electroferogram is an analysis of oligomaltose for use as a mobilitystandard. The bottom electroferogram is an analysis of RNAse B.

FIGS. 36A and 36B is the depiction of a nucleotide sequence of a cDNAencoding a mature form of Aspergillus niger (lacking signal peptide)glucosidase II α, which is codon-optimized cDNA for expression inYarrowia lipolytica. (SEQ ID NO:7).

FIG. 37 is the depiction of a nucleotide sequence of a cDNA encoding amature form of Aspergillus niger (lacking signal peptide) glucosidase IIβ, which is codon-optimized cDNA for expression in Yarrowia lipolytica.(SEQ ID NO:8).

FIG. 38 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from various Yarrowia lipolytica cells (Δalg3 andALG6 overexpressing along with the alpha subunit of glucosidase II fromAspergillus niger (An) expressed under the control of a TEF or hp4dpromoter) as indicated. The Y-axis represents the relative fluorescenceunits as an indication of the amount of each of the mannose structures.The X-axis represents the relative mobility of each complex mannosestructure through a gel. The top electroferogram is an analysis ofoligomaltose for use as a mobility standard. The bottom electroferogramis an analysis of RNAse B.

FIGS. 39A and 39B are a pair of bar graphs depicting the relativeexpression level (Y-axis) of the HAC1 (39A) or KAR (39B) gene in WT(MTLY60) Yarrowia lipolytica cells or in two clones (clone 7 and clone2) of Yarrowia lipolytica cells containing a spliced form of HAC1 cDNAunder the expression control of the hp4d promoter.

FIG. 40 is line graph depicting the growth of wild type Pichia pastorisGS115 cells transformed with an empty vector as compared to the growthof Pichia pastoris GS115 cells expressing the Hac1p protein.

FIG. 41 is a photograph of a Coomassie blue stained polyacrylamide gelcomparing the expression level of the murine IL-10 (mIL-10) protein froma culture of Pichia pastoris GS115 cell cells expressing mIL-10 proteinwith the expression of the mIL-10 protein obtained from a culture ofGS115 cells expressing mIL-10 and the spliced HAC1 protein from Pichiapastoris under the control of an inducible promoter, AOX1. The followingsamples were resolved in the gel: Lane 1 (“ladder”), a combination ofproteins of known molecular weight; Lane 2 (“Reference”), proteinobtained from the reference mIL-10 expressing Pichia pastoris strain(GS115); Lane 3 (“Reference”), protein obtained from the referencemIL-10 expressing Pichia pastoris strain after PNGase F enzyme treatmentof the proteins; Lane 4 (“Clone 1”), protein obtained from a mIL-10expressing Pichia pastoris cells inducibly expressing HAC1 protein; Lane5 (“Clone 1”), protein obtained from a mIL-10 expressing Pichia pastoriscells inducibly expressing HAC1 protein after treatment of the proteinwith PNGase F enzyme; Lane 6 (“Clone 2”), protein obtained from a mIL-10expressing Pichia pastoris cells inducibly expressing HAC1 protein 1;Lane 7 (“Clone 2”), protein obtained from a mIL-10 expressing Pichiapastoris cells inducibly expressing HAC1 protein after treatment of theproteins with PNGase F enzyme.

FIG. 42 is the depiction of a nucleotide sequence of an exemplary cDNAsequence encoding a Trichoderma reesei α-1,2 mannosidase, codonoptimized for expression in Yarrowia lipolytica (SEQ ID NO:9) containingthe LIP2 pre signal sequence.

FIG. 43 is the depiction of a nucleotide sequence of an exemplarynucleotide sequence for the GAP promoter of Yarrowia lipolytica. (SEQ IDNO:10).

FIGS. 44A-44C are the depiction of a nucleotide sequence of an exemplarynucleic acid sequence (SEQ ID NO:11) for the expression vectorpYLHUXdL2preManHDEL, which contains a cDNA sequence encoding aTrichoderma reesei α-1,2 mannosidase, codon optimized for expression inYarrowia lipolytica and containing the LIP2 pre signal sequence.

FIGS. 45A-45C are the depiction of a nucleotide sequence of an exemplarynucleic acid sequence (SEQ ID NO:12) for the expression vectorpYLGUXdL2preManHDEL, which contains a cDNA sequence encoding aTrichoderma reesei α-1,2 mannosidase, codon optimized for expression inYarrowia lipolytica and containing the LIP2 pre signal sequence.

FIGS. 46A-46C are the depiction of a nucleotide sequence of an exemplarynucleic acid sequence (SEQ ID NO:13) for the expression vectorpYLPUXdL2preManHDEL, which contains a cDNA sequence encoding aTrichoderma reesei α-1,2 mannosidase, codon optimized for expression inYarrowia lipolytica and containing the LIP2 pre signal sequence.

FIGS. 47A-47C are the depiction of a nucleotide sequence of an exemplarynucleic acid sequence (SEQ ID NO:14) for the expression vectorpYLTUXdL2preManHDEL, which contains a cDNA sequence encoding aTrichoderma reesei α-1,2 mannosidase, codon optimized for expression inYarrowia lipolytica and containing the LIP2 pre signal sequence.

FIG. 48 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from Yarrowia lipolytica cells transformed withdifferent expression vectors as indicated: “hp4dL2ManHDEL”(pYLHUXdL2preManHDEL, FIGS. 44A-44C); “GAPL2ManHDEL”(pYLGUXdL2preManHDEL, FIGS. 45A-45C); “TEF1L2ManHDEL”(pYLTUXdL2preManHDEL, FIGS. 47A-47C). The Y-axis represents the relativefluorescence units as an indication of the amount of each of the mannosestructures. The X-axis represents the relative mobility of each complexmannose structure through a gel. The top electroferogram is an analysisof dextran for use as a mobility standard. The second electroferogram inthe series is an analysis of RNAse B.

FIG. 49 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from Yarrowia lipolytica MTLY60 Δoch1 cellscontaining a stably integrated expression vector pYLTUXdL2preManHDEL(FIGS. 47A-47C). Glycoprotein samples were obtained from cell culturesat 24, 48, 72, and 96 hours. The top electroferogram is an analysis ofdextran for use as a mobility standard. The second electroferogram inthe series is an analysis of RNAse B.

FIG. 50 is an exemplary nucleic acid sequence for humanglucocerebrosidase (GLCM, Swiss Prot entry nr: P04062; SEQ ID NO:15),which was chemically synthesized as a codon-optimized cDNA forexpression in Yarrowia lipolytica.

FIG. 51 is a photograph of an immunoblot depicting the mobility patternof human glucocerebrosidase expressed in Yarrowia lipolytica strainsMTLY60 (WT; lanes 4 and 6) and MTLY60Δoch1 (Δoch1; first three lanes).The molecular weight (kDa) of the proteins is depicted, by way ofmolecular weight markers, at the far right of the immunoblot.

FIG. 52 is an exemplary nucleic acid sequence for human erythropoietin(Epo, Swiss Prot entry nr: P01588; SEQ ID NO:16), which was chemicallysynthesized as a codon-optimized cDNA for expression in Yarrowialipolytica.

FIG. 53 is an exemplary nucleic acid sequence for human α-galactosidaseA (AGAL, Swiss Prot entry nr: P06280; SEQ ID NO:17), which waschemically synthesized as a codon-optimized cDNA for expression inYarrowia lipolytica.

FIG. 54 is a series of electron micrographs of wild type Pichia pastoriscells or Pichia pastoris cells overexpressing the spliced form of Hac1pprotein. Discrete regions of stacked lipid membranes in the cells areboxed.

FIG. 55 is a series of electroferograms depicting N-glycan analysis ofglycoproteins obtained from WT Yarrowia lipolytica cells (poll d) andYarrowia lipolytica cells expressing a fusion protein ofalpha-1,2-mannosidase and a HDEL sequence as indicated. Analysis wasperformed using DSA-FACE. “M5,” “M6,” “M7,” “M8,” and “M9,” refer to thenumber of mannose residues conjugated to the chitobiose core structure.The Y-axis represents the relative fluorescence units as an indicationof the amount of each of the mannose structures. The X-axis representsthe relative mobility of each complex mannose structure through a gel.The top electroferogram is an analysis of RNAse B. The bottomelectroferogram is an analysis of oligomaltose for use as a mobilitystandard.

DETAILED DESCRIPTION

The methods and genetically engineered cells described herein can beused to produce target molecules (e.g., target protein or targetdolichol) having an altered N-glycosylation form as compared to theN-glycosylation form of the target molecules produced in non-geneticallyengineered cells. Administration of glycosylated target molecules (e.g.,glycosylated proteins) to patients having metabolic disorders (e.g.,lysosomal storage disorders) has been shown to ameliorate the symptomsof the disorders. Thus, the methods and cells described are useful forthe preparation of altered N-glycosylated target molecules for, interalia, the treatment of metabolic disorders such as lysosomal storagedisorders. Such altered N-glycosylation molecules are also useful in awide-variety of other fields, e.g., the food and beverage industries;the pharmaceutical industry (e.g., as vaccines); the agricultureindustry; and the chemical industry, to name a few.

Altered N-Glycosylation Molecule

Target molecules, as used herein, refer to any molecules that undergoaltered N-glycosylation by one or more N-glycosylation activities from agenetically engineered cell (e.g., a fungal cell such as Yarrowialipolytica or Arxula adeninivorans (or other related species dimorphicyeast) cell; a plant cell, or an animal cell). In some embodiments, thetarget molecules are capable of being trafficked through one or moresteps of the Yarrowia lipolytica or Arxula adeninivorans (or otherrelated species dimorphic yeast) secretory pathway, resulting in theiraltered N-glycosylation by the host cell machinery. The target moleculescan be endogenous or exogenous.

Target proteins, their biologically active fragments, or biologicallyactive variants thereof, can include proteins containing additions,deletions, or substitutions as described above. Suitable target proteinsinclude pathogen proteins (e.g., tetanus toxoid; diptheria toxoid; viralsurface proteins (e.g., cytomegalovirus (CMV) glycoproteins B, H andgCIII; human immunodeficiency virus 1 (HIV-1) envelope glycoproteins;Rous sarcoma virus (RSV) envelope glycoproteins; herpes simplex virus(HSV) envelope glycoproteins; Epstein Barr virus (EBV) envelopeglycoproteins; varicella-zoster virus (VZV) envelope glycoproteins;human papilloma virus (HPV) envelope glycoproteins; Influenza virusglycoproteins; and Hepatitis family surface antigens), lysosomalproteins (e.g., glucocerebrosidase, cerebrosidase, orgalactocerebrosidase), insulin, glucagon, growth factors, cytokines,chemokines, antibodies or fragments thereof, or fusions of any of theproteins to antibodies or fragments of antibodies (e.g., protein-Fc).Growth factors include, e.g., vascular endothelial growth factor (VEGF),Insulin-like growth factor (IGF), bone morphogenic protein (BMP),Granulocyte-colony stimulating factor (G-CSF), Granulocyte-macrophagecolony stimulating factor (GM-CSF), Nerve growth factor (NGF); aNeurotrophin, Platelet-derived growth factor (PDGF), Erythropoietin(EPO), Thrombopoietin (TPO), Myostatin (GDF-8), Growth Differentiationfactor-9 (GDF9), basic fibroblast growth factor (bFGF or FGF2),Epidermal growth factor (EGF), Hepatocyte growth factor (HGF). Cytokinesinclude, e.g., interleukins (e.g., IL-1 to IL-33 (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, orIL-15)). Chemokines include, e.g., 1-309, TCA-3, MCP-1, MIP-1α, MIP-1β,RANTES, C10, MRP-2, MARC, MCP-3, MCP-2, MRP-2, CCF18, MIP-1γ, Eotaxin,MCP-5, MCP-4, NCC-1, CkIβ10, HCC-1, Leukotactin-1, LEC, NCC-4, TARC,PARC, or Eotaxin-2. Also included are tumor glycoproteins (e.g.,tumor-associated antigens), for example, carcinoembryonic antigen (CEA),human mucins, HER-2/neu, and prostate-specific antigen (PSA) [R. A.Henderson and O. J. Finn, Advances in Immunology, 62, pp. 217-56(1996)]. In some embodiments, the target protein can be one associatedwith a lysosomal storage disorder, which target proteins include, e.g.,alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase,beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase,arylsulfatase B, arylsulfatase A, alpha-N-acetylgalactosaminidase,aspartylglucosaminidase, iduronate-2-sulfatase,alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase,hyaluronidase, alpha-L-mannosidase, alpha-neuraminidase,phosphotransferase, acid lipase, acid ceramidase, sphingomyelinase,thioesterase, cathepsin K, and lipoprotein lipase.

Target proteins can also be fusion proteins. Fusions proteins include,e.g., a fusion of (i) any protein described herein or fragment thereofwith (ii) an antibody or fragment thereof. As used herein, the term“antibody fragment” refers to an antigen-binding fragment, e.g., Fab,F(ab′)₂, Fv, and single chain Fv (scFv) fragments. An scFv fragment is asingle polypeptide chain that includes both the heavy and light chainvariable regions of the antibody from which the scFv is derived. Inaddition, diabodies [Poljak (1994) Structure 2(12):1121-1123; Hudson etal. (1999) J. Immunol. Methods 23(1-2):177-189, the disclosures of bothof which are incorporated herein by reference in their entirety] andintrabodies [Huston et al. (2001) Hum. Antibodies 10(3-4):127-142;Wheeler et al. (2003) Mol. Ther. 8(3):355-366; Stocks (2004) DrugDiscov. Today 9(22): 960-966, the disclosures of all of which areincorporated herein by reference in their entirety] can be used in themethods of the invention.

Target proteins can also be joined to one or more of a polymer, acarrier, an adjuvant, an immunotoxin, or a detectable (e.g.,fluorescent, luminescent, or radioactive) moiety. For example, a targetprotein can be joined to polyethyleneglycol, which polymer moiety can beused, e.g., to increase the molecular weight of small proteins and/orincrease circulation residence time.

In some embodiments, the target molecule can be, or contain, dolichol.

Genetically Engineered Cells

Described herein are genetically engineered cells having at least onemodified N-glycosylation activity, which cells are useful for theproduction of one or more target molecules having an alteredN-glycosylation form. Cells suitable for genetic engineering include,e.g., fungal cells (e.g., Yarrowia lipolytica or any other relateddimorphic yeast cells described herein), plant cells, or animal cells(e.g., (nematode, insect, plant, bird, reptile, or mammal (e.g., amouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse,whale, monkey, or human)). The cells can be primary cells, immortalizedcells, or transformed cells. The cells can be those in an animal, e.g.,a non-human mammal. Such cells, prior to the genetic engineering asspecified herein, can be obtained from a variety of commercial sourcesand research resource facilities, such as, for example, the AmericanType Culture Collection (Rockville, Md.). Target molecules includeproteins such as any of the target proteins described herein (seeabove). Target molecules also include dolichol.

Genetic engineering of a cell includes genetic modifications such as:(i) deletion of an endogenous gene encoding a protein havingN-glycosylation activity; (ii) introduction of a recombinant nucleicacid encoding a mutant form of a protein (e.g., endogenous or exogenousprotein) having N-glycosylation activity (i.e., expressing a mutantprotein having an N-glycosylation activity); (iii) introduction orexpression of an RNA molecule that interferes with the functionalexpression of a protein having the N-glycosylation activity; (iv)introduction of a recombinant nucleic acid encoding a wild-type (e.g.,endogenous or exogenous) protein having N-glycosylation activity (i.e.,expressing a protein having an N-glycosylation activity); or (v)altering the promoter or enhancer elements of one or more endogenousgenes encoding proteins having N-glycosylation activity to thus alterthe expression of their encoded proteins. RNA molecules include, e.g.,small-interfering RNA (siRNA), short hairpin RNA (shRNA), anti-senseRNA, or micro RNA (miRNA). It is understood that item (ii) includes,e.g., replacement of an endogenous gene (e.g., by homologousrecombination) with a gene encoding a protein having greaterN-glycosylation activity relative to the endogenous gene so replaced.Genetic engineering also includes altering an endogenous gene encoding aprotein having an N-glycosylation activity to produce a protein havingadditions (e.g., a heterologous sequence), deletions, or substitutions(e.g., mutations such as point mutations; conservative ornon-conservative mutations). Mutations can be introduced specifically(e.g., site-directed mutagenesis or homologous recombination; seeaccompanying Examples) or can be introduced randomly (for example, cellscan be chemically mutagenized as described in, e.g., Newman andFerro-Novick (1987) J. Cell Biol. 105(4):1587, the disclosure of whichis incorporated herein by reference in its entirety.

The genetic modifications described herein can result in one or more of(i) an increase in one or more N-glycosylation activities in thegenetically modified cell, (ii) a decrease in one or moreN-glycosylation activities in the genetically modified cell, (iii) achange in the localization or intracellular distribution of one or moreN-glycosylation activities in the genetically modified cell, or (iv) achange in the ratio of one or more N-glycosylation activities in thegenetically modified cell. It is understood that an increase in theamount of an N-glycosylation activity can be due to overexpression ofone or more proteins having N-glycosylation activity, an increase incopy number of an endogenous gene (e.g., gene duplication), or analteration in the promoter or enhancer of an endogenous gene thatstimulates an increase in expression of the protein encoded by the gene.A decrease in one or more N-glycosylation activities can be due tooverexpression of a mutant form (e.g., a dominant negative form) of oneor more proteins having N-glysosylation altering activities,introduction or expression of one or more interfering RNA molecules thatreduce the expression of one or more proteins having an N-glycosylationactivity, or deletion of one or more endogenous genes that encode aprotein having N-glycosylation activity.

Methods of deleting or disrupting one or more endogenous genes aredescribed in the accompanying Examples. For example, to disrupt a geneby homologous recombination, a “gene replacement” vector can beconstructed in such a way to include a selectable marker gene. Theselectable marker gene can be operably linked, at both 5′ and 3′ end, toportions of the gene of sufficient length to mediate homologousrecombination. The selectable marker can be one of any number of geneswhich either complement host cell auxotrophy or provide antibioticresistance, including URA3, LEU2 and HIS3 genes. Other suitableselectable markers include the CAT gene, which confers chloramphenicolresistance to yeast cells, or the lacZ gene, which results in bluecolonies due to the expression of β-galactosidase. Linearized DNAfragments of the gene replacement vector are then introduced into thecells using methods well known in the art (see below). Integration ofthe linear fragments into the genome and the disruption of the gene canbe determined based on the selection marker and can be verified by, forexample, Southern blot analysis.

As detailed in the accompanying examples, subsequent to its use inselection, a selectable marker can be removed from the genome of thehost cell by, e.g., Cre-loxP systems (see below).

Alternatively, a gene replacement vector can be constructed in such away as to include a portion of the gene to be disrupted, which portionis devoid of any endogenous gene promoter sequence and encodes none oran inactive fragment of the coding sequence of the gene. An “inactivefragment” is a fragment of the gene that encodes a protein having, e.g.,less than about 10% (e.g., less than about 9%, less than about 8%, lessthan about 7%, less than about 6%, less than about 5%, less than about4%, less than about 3%, less than about 2%, less than about 1%, or 0%)of the activity of the protein produced from the full-length codingsequence of the gene. Such a portion of the gene is inserted in a vectorin such a way that no known promoter sequence is operably linked to thegene sequence, but that a stop codon and a transcription terminationsequence are operably linked to the portion of the gene sequence. Thisvector can be subsequently linearized in the portion of the genesequence and transformed into a cell. By way of single homologousrecombination, this linearized vector is then integrated in theendogenous counterpart of the gene.

Expression vectors can be autonomous or integrative.

A recombinant nucleic acid (e.g., one encoding a wild-type or mutantform of a protein having N-glycosylation activity) can be in introducedinto the cell in the form of an expression vector such as a plasmid,phage, transposon, cosmid or virus particle. The recombinant nucleicacid can be maintained extrachromosomally or it can be integrated intothe yeast cell chromosomal DNA. Expression vectors can contain selectionmarker genes encoding proteins required for cell viability underselected conditions (e.g., URA3, which encodes an enzyme necessary foruracil biosynthesis or TRP1, which encodes an enzyme required fortryptophan biosynthesis) to permit detection and/or selection of thosecells transformed with the desired nucleic acids (see, e.g., U.S. Pat.No. 4,704,362). Expression vectors can also include an autonomousreplication sequence (ARS). For example, U.S. Pat. No. 4,837,148describes autonomous replication sequences which provide a suitablemeans for maintaining plasmids in Pichia pastoris. The disclosure ofU.S. Pat. No. 4,837,148 is incorporated herein by reference in itsentirety.

Integrative vectors are disclosed, e.g., in U.S. Pat. No. 4,882,279 (thedisclosure of which is incorporated herein by reference in itsentirety). Integrative vectors generally include a serially arrangedsequence of at least a first insertable DNA fragment, a selectablemarker gene, and a second insertable DNA fragment. The first and secondinsertable DNA fragments are each about 200 (e.g., about 250, about 300,about 350, about 400, about 450, about 500, or about 1000 or more)nucleotides in length and have nucleotide sequences which are homologousto portions of the genomic DNA of the species to be transformed. Anucleotide sequence containing a gene of interest (e.g., a gene encodinga protein having N-glycosylation activity) for expression is inserted inthis vector between the first and second insertable DNA fragmentswhether before or after the marker gene. Integrative vectors can belinearized prior to yeast transformation to facilitate the integrationof the nucleotide sequence of interest into the host cell genome.

An expression vector can feature a recombinant nucleic acid under thecontrol of a yeast (e.g., Yarrowia lipolytica, Arxula adeninivorans, orother related dimorphic yeast species) promoter, which enables them tobe expressed in yeast. Suitable yeast promoters include, e.g., ADC1,TPI1, ADH2, hp4d, PDX, and Gall° (see, e.g., Guarente et al. (1982)Proc. Natl. Acad. Sci. USA 79(23):7410) promoters. Additional suitablepromoters are described in, e.g., Zhu and Zhang (1999) Bioinformatics15(7-8):608-611 and U.S. Pat. No. 6,265,185, the disclosures of each ofwhich are incorporated herein by reference in their entirety. Where theexpression vector is to be introduced into an animal cell, such as amammalian cell, the expression vector can feature a recombinant nucleicacid under the control of an animal cell promoter suitable forexpression in the host cell of interest. Examples of mammalian promotersinclude, e.g., SV40 or cytomegalovirus (CMV) promoters.

A promoter can be constitutive or inducible (conditional). Aconstitutive promoter is understood to be a promoter whose expression isconstant under the standard culturing conditions. Inducible promotersare promoters that are responsive to one or more induction cues. Forexample, an inducible promoter can be chemically regulated (e.g., apromoter whose transcriptional activity is regulated by the presence orabsence of a chemical inducing agent such as an alcohol, tetracycline, asteroid, a metal, or other small molecule) or physically regulated(e.g., a promoter whose transcriptional activity is regulated by thepresence or absence of a physical inducer such as light or high or lowtemperatures). An inducible promoter can also be indirectly regulated byone or more transcription factors that are themselves directly regulatedby chemical or physical cues.

Genetic engineering of a cell also includes activating an endogenousgene (e.g., a gene encoding a protein having N-glycosylation activity)that is present in the host cell, but is normally not expressed in thecells or is not expressed at significant levels in the cells. Forexample, a regulatory sequence (e.g., a gene promoter or an enhancer) ofa endogenous gene can be modified such that the operably-linked codingsequence exhibits increased expression. Homologous recombination ortargeting can be used to replace or disable the regulatory regionnormally associated with the gene with a regulatory sequence whichcauses the gene to be expressed at levels higher than evident in thecorresponding non-genetically engineered cell, or causes the gene todisplay a pattern of regulation or induction that is different thanevident in the corresponding non-genetically engineered cell. Suitablemethods for introducing alterations of a regulatory sequence (e.g., apromoter or enhancer) of a gene are described in, e.g., U.S. ApplicationPublication No. 20030147868, the disclosure of which is incorporatedherein by reference in its entirety.

It is understood that other genetically engineered modifications canalso be conditional. For example, a gene can be conditionally deletedusing, e.g., a site-specific DNA recombinase such as the Cre-loxP system(see, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173 and U.S.Application Publication No. 20060014264, the disclosures of each ofwhich are incorporated by reference in their entirety).

A recombinant nucleic acid can be introduced into a cell describedherein using a variety of methods such as the spheroplast technique orthe whole-cell lithium chloride yeast transformation method. Othermethods useful for transformation of plasmids or linear nucleic acidvectors into cells are described in, for example, U.S. Pat. No.4,929,555; Hinnen et al. (1978) Proc. Nat. Acad. Sci. USA 75:1929; Itoet al. (1983) J. Bacteriol. 153:163; U.S. Pat. No. 4,879,231; andSreekrishna et al. (1987) Gene 59:115, the disclosures of each of whichare incorporated herein by reference in their entirety. Electroporationand PEG1000 whole cell transformation procedures may also be used, asdescribed by Cregg and Russel, Methods in Molecular Biology: PichiaProtocols, Chapter 3, Humana Press, Totowa, N.J., pp. 27-39 (1998), thedisclosure of which is incorporated herein by reference in its entirety.Transfection of animal cells can feature, for example, the introductionof a vector to the cells using calcium phosphate, electroporation, heatshock, liposomes, or transfection reagents such as FUGENE® orLIPOFECTAMINE®, or by contacting naked nucleic acid vectors with thecells in solution (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y., USA, November 1989; thedisclosure of which is incorporated herein by reference in itsentirety).

Transformed yeast cells can be selected for by using appropriatetechniques including, but not limited to, culturing auxotrophic cellsafter transformation in the absence of the biochemical product required(due to the cell's auxotrophy), selection for and detection of a newphenotype, or culturing in the presence of an antibiotic which is toxicto the yeast in the absence of a resistance gene contained in thetransformants. Transformants can also be selected and/or verified byintegration of the expression cassette into the genome, which can beassessed by, e.g., Southern blot or PCR analysis.

Prior to introducing the vectors into a target cell of interest, thevectors can be grown (e.g., amplified) in bacterial cells such asEscherichia coli (E. coli). The vector DNA can be isolated frombacterial cells by any of the methods known in the art which result inthe purification of vector DNA from the bacterial milieu. The purifiedvector DNA can be extracted extensively with phenol, chloroform, andether, to ensure that no E. coli proteins are present in the plasmid DNApreparation, since these proteins can be toxic to mammalian cells.

Genetic engineering, as described herein, can be used to express (e.g.,overexpress), introduce modifications into, or delete any number ofgenes, e.g., genes encoding proteins having N-glycosylation activity.Such genes include, e.g., ALG7, ALG13, ALG14, ALG1, ALG2, ALG11, RFT1,ALG3, ALG9, ALG12, ALG6, ALG8, ANL1, ALG10, ALG5, OST3, OST4, OST6,STT3, OST1, OST5, WBP1, SWP1, OST2, DPM1, SEC59, OCH1, MNN9, VAN1, MNN8,MNN10, MNN11, HOC1, MNN2, MNN5, MNN6, KTR1, YUR1, MNN4, KRE2, KTR2,KTR3, MNN1, MNS1, PNO1, MNN9, glucosidase I, glucosidase II, orendomannosidase. The genes encoding proteins having N-glycosylationactivity can be from any species (e.g., lower eukaryotes (e.g., fungus(including yeasts) or trypanosomes), plant, or animal (e.g., insect,bird, reptile, or mammal (e.g., a rodent such as mouse or rat, dog, cat,horse, goat, cow, pig, non-human primate, or human)) containing suchgenes. Exemplary fungal species from which genes encoding proteinshaving N-glycosylation activity can be obtained include, withoutlimitation, Pichia anomala, Pichia bovis, Pichia canadensis, Pichiacarsonii, Pichia farinose, Pichia fermentans, Pichia fluxuum, Pichiamembranaefaciens, Pichia membranaefaciens, Candida valida, Candidaalbicans, Candida ascalaphidarum, Candida amphixiae, Candida Antarctica,Candida atlantica, Candida atmosphaerica, Candida blattae, Candidacarpophila, Candida cerambycidarum, Candida chauliodes, Candidacorydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis,Candida fructus, Candida glabrata, Candida fermentati, Candidaguilliermondii, Candida haemulonii, Candida insectamens, Candidainsectorum, Candida intermedia, Candida jeffresii, Candida kefyr,Candida krusei, Candida lusitaniae, Candida lyxosophila, Candidamaltosa, Candida membranifaciens, Candida milleri, Candida oleophila,Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candidashehatea, Candida temnochilae, Candida tenuis, Candida tropicalis,Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candidaviswanathii, Candida utilis, Pichia membranaefaciens, Pichia silvestris,Pichia membranaefaciens, Pichia chodati, Pichia membranaefaciens, Pichiamenbranaefaciens, Pichia minuscule, Pichia pastoris, Pichiapseudopolymorpha, Pichia quercuum, Pichia robertsii, Pichia saitoi,Pichia silvestrisi, Pichia strasburgensis, Pichia terricola, Pichiavanriji, Pseudozyma Antarctica, Rhodosporidium toruloides, Rhodotorulaglutinis, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomycesmomdshuricus, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomycescerevisiae, Saccharomyces bisporus, Saccharomyces chevalieri,Saccharomyces delbrueckii, Saccharomyces exiguous, Saccharomycesfermentati, Saccharomyces fragilis, Saccharomyces marxianus,Saccharomyces mellis, Saccharomyces rosei, Saccharomyces rouxii,Saccharomyces uvarum, Saccharomyces willianus, Saccharomycodes ludwigii,Saccharomycopsis capsularis, Saccharomycopsis fibuligera,Saccharomycopsis fibuligera, Endomyces hordei, Endomycopsis fobuligera.Saturnispora saitoi, Schizosaccharomyces octosporus, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporadelbrueckii, Saccharomyces dairensis, Torulaspora delbrueckii,Torulaspora fermentati, Saccharomyces fermentati, Torulasporadelbrueckii, Torulaspora rosei, Saccharomyces rosei,Torulasporadelbrueckii, Saccharomyces rosei, Torulaspora delbrueckii, Saccharomycesdelbrueckii, Torulaspora delbrueckii, Saccharomyces delbrueckii,Zygosaccharomyces mongolicus, Dorulaspora globosa, Debaryomycesglobosus, Torulopsis globosa, Trichosporon cutaneum, Trigonopsisvariabilis, Williopsis californica, Williopsis saturnus,Zygosaccharomyces bisporus, Zygosaccharomyces bisporus, Debaryomycesdisporua. Saccharomyces bisporas, Zygosaccharomyces bisporus,Saccharomyces bisporus, Zygosaccharomyces mellis, Zygosaccharomycespriorianus, Zygosaccharomyces rouxiim, Zygosaccharomyces rouxii,Zygosaccharomyces barkeri, Saccharomyces rouxii, Zygosaccharomycesrouxii, Zygosaccharomyces major, Saccharomyces rousii, Pichia anomala,Pichia bovis, Pichia Canadensis, Pichia carsonii, Pichia farinose,Pichia fermentans, Pichia fluxuum, Pichia membranaefaciens, Pichiapseudopolymorpha, Pichia quercuum, Pichia robertsii, PseudozymaAntarctica, Rhodosporidium toruloides, Rhodosporidium toruloides,Rhodotorula glutinis, Saccharomyces bayanus, Saccharomyces bayanus,Saccharomyces bisporus, Saccharomyces cerevisiae, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces fermentati,Saccharomyces fragilis, Saccharomycodes ludwigii, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporaglobosa, Trigonopsis variabilis, Williopsis californica, Williopsissaturnus, Zygosaccharomyces bisporus, Zygosaccharomyces mellis,Zygosaccharomyces rouxii, or any other fungi (e.g., yeast) known in theart or described herein. Exemplary lower eukaryotes also include variousspecies of Aspergillus including, but not limited to, Aspergilluscaesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillusclavatus, Aspergillus deflectus, Aspergillus flavus, Aspergillusfumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger,Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus,Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae,Aspergillus sydowi, Aspergillus tamari, Aspergillus terreus, Aspergillusustus, or Aspergillus versicolor. Exemplary protozoal genera from whichgenes encoding proteins having N-glycosylation activity can be obtainedinclude, without limitation, Blastocrithidia, Crithidia, Endotrypanum,Herpetomonas, Leishmania, Leptomonas, Phytomonas, Trypanosoma (e.g., T.bruceii, T. gambiense, T. rhodesiense, and T. Cruzi), and Wallaceina.

It is understood that genetic engineering, as described herein, can beused to express (e.g., overexpress), introduce modifications into, ordelete any number of genes (e.g., genes encoding proteins havingN-glycosylation activity) and95/or any combination of one or more (e.g.,two, three, four, five, six, seven, eight, nine, 10, 11, 12, 15, or 20or more) of any of the genes recited herein.

In some embodiments, the genetically engineered cell lacks the ALG3(Genbank®Accession Nos: XM_(—)503488, Genolevures Ref: YALI0E03190g)gene or gene product (e.g., mRNA or protein) thereof. In someembodiments, the genetically engineered cell expresses (e.g.,overexpresses) the ALG6 (Genbank® Acccession Nos: XM_(—)502922,Genolevures Ref: YALI0D17028g) protein. In some embodiments, thegenetically engineered cell expresses the MNN4 gene (Genbank® AcccessionNos: XM_(—)503217, Genolevures Ref: YALI0D24101g). In some embodiments,the genetically engineered cell lacks the OCH1 and/or MNN9 gene or geneproducts (e.g., mRNA or protein) thereof. In some embodiments, thegenetically engineered cell does not lack the OCH1 gene or a geneproduct (e.g., mRNA or protein) thereof. In some embodiments, thegenetically engineered cell expresses an alpha or beta subunit (or boththe alpha and the beta subunit) of a glucosidase II such as theglucosidase II of Yarrowia lipolytica or Trypanosoma brucei. In someembodiments, the genetically engineered cell expresses a mutantase suchas the mutanase of T. harzianum. In some embodiments, the geneticallyengineered cell can have any combination of these modifications.

For example, in some embodiments, the genetically engineered cell canlack the ALG3 (e.g., the ALG3 gene exemplified by Genbank® AccessionNos: XM_(—)503488, Genolevures Ref: YALI0E03190g) gene or gene product(e.g., mRNA or protein) thereof; can overexpress the ALG6 (e.g., theALG6 as exemplified by Genbank® Acccession Nos: XM_(—)502922,Genolevures Ref: YALI0D17028g) protein; can overexpress one or both ofthe alpha and the beta subunit of a glucosidase II (such as theglucosidase II of Yarrowia lipolytica, Trypanosoma brucei, or any otherspecies described herein); can overexpress an alpha-1,2-mannosidase; andoverexpress one or more (and any combination) of the following: aglycosidase, a glycosyltransferase, a sugar-nucleotide transporter, asugar-nucleotide modifying enzyme. In some embodiments, the geneticallyengineered cell does not lack the OCH1 gene or a gene product (e.g.,mRNA or protein) thereof.

In some embodiments, the genetically modified cell can contain amannosidase activity (e.g., an α-mannosidase activity). The mannosidaseactivity can be targeted to the endoplasmic reticulum. The mannosidasecan have a pH optimum at least below 7.5 (e.g., at least below 7.4, atleast below 7.3, at least below 7.2, at least below 7.1, at least below7.0, at least below 6.9, at least below 6.8, at least 6.7, at leastbelow 6.6, at least below 6.5, at least 6.4, at least below 6.3, atleast below 6.2, at least below 6.1, at least below 6.0, at least below5.9, at least below 5.8, at least below 5.7, at least below 5.6, atleast below 5.5, at least below 5.4, at least below 5.3, at least below5.2, at least below 5.1, at least below 5.0, at least below 4.9, atleast below 4.8, or at least below 4.7).

The mannosidase can be MNS1.

For example, the genetically engineered cell can overexpress amannosidase (e.g., an alpha-1,2-mannosidase or any other mannosidasedescribed herein), but not lack the OCH1 gene or a gene product (e.g.,mRNA or protein) thereof. The mannosidase can be a wild-type form of theprotein or can be a mutant form such as a fusion protein containing amannosidase and an HDEL ER-retention amino acid sequence (see Examples).(It is understood that any protein having N-glycosylation activity canbe engineered into a fusion protein comprising an HDEL sequence).

In some embodiments, the genetically modified cell can contain anactivity capable of promoting mannosyl phosphorylation of the alteredN-glycosylation form of the target molecule. For example, a nucleic acidencoding an activity that promotes phosphorylation of N-glycans (e.g.MNN4, MNN6, PNO1) can be introduced in the genetically engineered cell,which cell is capable of increasing phosphorylating the N-glycosylationof the target molecule.

In some embodiments, the genetically modified cell can contain anactivity capable of removing mannose residues that cap phosphorylation(e.g., a mannosidase such as the one from Jack Bean) from the alteredN-glycosylation molecules.

In some embodiments, the genetically modified cell is capable ofremoving glucose residues from Man₅GlcNAc₂. For example, the geneticallymodified cell can overexpress a protein having α-1,3-glucosidaseactivity such as, but not limited to, a mutanase or one or both of thealpha and beta subunit of a glucosidase II (such as the glucosidase IIof Yarrowia lipolytica, Trypanosoma brucei, or any other fungal speciesdescribed herein).

In embodiments where a protein having N-glycosylation activity isderived from a cell that is of a different type (e.g., of a differentspecies) than the cell into which the protein is to be expressed, anucleic acid encoding the protein can be codon-optimized for expressionin the particular cell of interest. For example, a nucleic acid encodinga protein having N-glycosylation from Trypanosoma brucei can becodon-optimized for expression in a yeast cell such as Yarrowialipolytica. Such codon-optimization can be useful for increasingexpression of the protein in the cell of interest. Methods forcodon-optimizing a nucleic acid encoding a protein are known in the artand described in, e.g., Gao et al. (Biotechnol. Prog. (2004) 20(2):443-448), Kotula et al. (Nat. Biotechn. (1991) 9, 1386-1389), andBennetzen et al. (J. Biol. Chem. (1982) 257(6):2036-3031).

A cell can also be genetically engineered to produce predominantlyN-glycans that are intermediates of a mammalian (e.g., human)glycosylation pathway. For example, one or more nucleic acids encodinghuman proteins having N-glycosylation activity can be introduced intothe cell. In some embodiments, human proteins can be introduced into thecell and one or more endogenous yeast proteins having N-glycosylationactivity can be suppressed (e.g., deleted or mutated). Techniques for“humanizing” a fungal glycosylation pathway are described in, e.g., Choiet al. (2003) Proc. Natl. Acad. Sci. USA 100(9):5022-5027; Verveken etal. (2004) Appl. Environ. Microb. 70(5):2639-2646; and Gerngross (2004)Nature Biotech. 22(11):1410-1414, the disclosures of each of which areincorporated herein by reference in their entirety.

Where the genetic engineering involves, e.g., changes in the expressionof a protein or expression of an exogenous protein (including a mutantform of an endogenous protein), a variety of techniques can be used todetermine if the genetically engineered cells express the protein. Forexample, the presence of mRNA encoding the protein or the protein itselfcan be detected using, e.g., Northern Blot or RT-PCR analysis or WesternBlot analysis, respectively. The intracellular localization of a proteinhaving N-glycosylation activity can be analyzed by using a variety oftechniques, including subcellular fractionation and immunofluorescence.

Additional genetic modifications and methods for introducing them intoany of the cells described herein can be adapted from the disclosuresof, e.g., U.S. Pat. Nos. 7,029,872; 5,272,070; and 6,803,225; and U.S.Application Publication Nos. 20050265988, 20050064539, 20050170452, and20040018588, the disclosures of each of which are incorporated herein byreference in their entirety.

While the engineering steps performed in dimorphic yeast species toachieve in vivo production of the Man₅GlcNAc₂ and Man₃GlcNAc₂ can bedifferent from the engineering steps performed in other yeast species,it will be clear to those skilled in the art that the engineeringtechniques to produce modified glycoproteins (with the Man₅GlcNAc₂ andMan₃GlcNAc₂ core N-glycan structures) in dimorphic yeasts in vivo can beadapted by routine experimentation from the methods disclosed in, interalia, U.S. Pat. No. 7,326,681 and U.S. Publication Nos. 20040018590,20060040353, and 20060286637 (the disclosure of each of which isincorporated by reference in its entirety). The adapted methods can thusbe used to achieve production of glycoproteins modified with human-typehybrid and complex N-glycans. These complex N-glycans can have 2 to 5branches initiated with a GlcNAc residue onto the above-named coreglycans, which can be further extended, e.g., with galactose, fucose andsialic acid residues.

In some embodiments, the mutant or wild-type proteins havingN-glycosylation activity can be isolated from the genetically engineeredcells using standard techniques. For example, following the expressionof a mutant or wild-type protein in the genetically engineered cell, theprotein can be isolated from the cell itself or from the media in whichthe cell was cultured. Methods of isolating proteins are known in theart and include, e.g., liquid chromatography (e.g., HPLC), affinitychromatography (e.g., metal chelation or immunoaffinity chromatography),ion-exchange chromatography, hydrophobic-interaction chromatography,precipitation, or differential solubilization.

In some embodiments, the isolated proteins having N-glycosylationactivity can be frozen, lyophilized, or immobilized and stored underappropriate conditions, which allow the proteins to retain activity.

The disclosure also provides a substantially pure culture of any of thegenetically engineered cells described herein. As used herein, a“substantially pure culture” of a genetically engineered cell is aculture of that cell in which less than about 40% (i.e., less thanabout: 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%;0.01%; 0.001%; 0.0001%; or even less) of the total number of viablecells in the culture are viable cells other than the geneticallyengineered cell, e.g., bacterial, fungal (including yeast), mycoplasmal,or protozoan cells. The term “about” in this context means that therelevant percentage can be 15% percent of the specified percentage aboveor below the specified percentage. Thus, for example, about 20% can be17% to 23%. Such a culture of genetically engineered cells includes thecells and a growth, storage, or transport medium. Media can be liquid,semi-solid (e.g., gelatinous media), or frozen. The culture includes thecells growing in the liquid or in/on the semi-solid medium or beingstored or transported in a storage or transport medium, including afrozen storage or transport medium. The cultures are in a culture vesselor storage vessel or substrate (e.g., a culture dish, flask, or tube ora storage vial or tube).

The genetically engineered cells described herein can be stored, forexample, as frozen cell suspensions, e.g., in buffer containing acryoprotectant such as glycerol or sucrose, as lyophilized cells.Alternatively, they can be stored, for example, as dried cellpreparations obtained, e.g., by fluidized bed drying or spray drying, orany other suitable drying method.

Methods of Producing Altered N-Glycosylation Molecules

Described herein are methods of producing an altered N-glycosylationform of a target molecule. The methods generally involve the step ofcontacting a target molecule with one or more N-glycosylation activitiesfrom a genetically engineered cell (e.g., a fungal cell (e.g., Yarrowialipolytica, Arxula adeninivorans, or any other related dimorphic yeastcells described herein), a plant cell, or an animal cell (e.g.,nematode, insect, plant, bird, reptile, or mammal (e.g., a mouse, rat,rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey,or human)). The methods can be cell-based or non-cell based.

Cell based methods can include the steps of introducing into a cell(e.g., a fungal cell (e.g., Yarrowia lipolytica, Arxula adeninivorans,or any other related dimorphic yeast cells described herein), a plantcell, or an animal cell) genetically engineered to have at least onemodified N-glycosylation activity a nucleic acid encoding a targetmolecule subject to N-glycosylation in the cell, wherein the cellproduces the target molecule in an altered N-glycosylation form. Thetarget molecule can be, e.g., a protein such as any of the targetproteins described herein. In embodiments where the target protein is alipid, the nucleic acid can be one encoding one or more enzymes whichpromote the synthesis of the lipid.

The types of modifications produced by the genetic engineering of thecells are described herein (see the accompanying Examples and“Genetically Engineered Cells” above).

Methods for introducing a nucleic acid are known in the art and aredescribed in the accompanying Examples and above.

Introduction or expression of a target molecule (e.g., a target protein)into a genetically engineered cell can result in the trafficking of thetarget molecule through the endoplasmic reticulum and/or Golgi apparatusof the cell, thereby producing an altered N-glycosylation form of thetarget molecule.

Following the processing of the target molecule (e.g., in thegenetically modified cell), the altered N-glycosylation form of thetarget molecule (e.g., the target protein) can contain one or moreN-glycan structures. For example, the altered form of the targetmolecule can contain one or more specific N-glycan structures such asMan₅GlcNAc₂ (structural formula I or VII; FIG. 4A), Man₈GlcNAc₂(structural formula I; FIG. 4A), Man₉GlcNAc₂ (structural formula II;FIG. 4A), Man₃GlcNAc₂ (structural formula XIV; FIG. 4B), Glc₁Man₅GlcNAc₂(structural formula VIII; FIG. 4B), or Glc₂Man₅GlcNAc₂ (structuralformula IX; FIG. 4B) (“Man” is mannose; “Glc” is glucose; and “GlcNAc”is N-acetylglucosamine).

The target molecules having altered N-glycosylation produced from thegenetically engineered cells can be homogeneous (i.e., all alteredN-glycosylation molecules containing the same specific N-glycanstructure) or can be substantially homogeneous. By “substantiallyhomogeneous” is meant that the altered target molecules are at leastabout 25% (e.g., at least about 27%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, or at least about 95%, or at least about 99%) of thetarget molecules having altered N-glycosylation produced by thegenetically engineered cell.

Where the genetically engineered cell includes one or moreN-glycosylation activities that effect the phosphorylation of anN-glycan, an altered N-glycosylation form of a target molecule can haveat least about 25% (e.g., at least about 27%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, or at least about 80%) of itsmannosyl residues phosphorylated.

Where any of the genetic modifications of the genetically engineeredcell are inducible or conditional on the presence of an inducing cue(e.g., a chemical or physical cue), the genetically engineered cell can,optionally, be cultured in the presence of an inducing agent before,during, or subsequent to the introduction of the nucleic acid. Forexample, following introduction of the nucleic acid encoding a targetprotein, the cell can be exposed to a chemical inducing agent that iscapable of promoting the expression of one or more proteins havingN-glycosylation activity. Where multiple inducing cues induceconditional expression of one or more proteins having N-glycosylationactivity, a cell can be contacted with multiple inducing agents.

Following processing by one or more N-glycosylation activities, thealtered target molecule can be isolated. The altered target molecule canbe maintained within the yeast cell and released upon cell lysis or thealtered target molecule can be secreted into the culture medium via amechanism provided by a coding sequence (either native to the exogenousnucleic acid or engineered into the expression vector), which directssecretion of the molecule from the cell. The presence of the alteredtarget molecule in the cell lysate or culture medium can be verified bya variety of standard protocols for detecting the presence of themolecule. For example, where the altered target molecule is a protein,such protocols can include, but are not limited to, immunoblotting orradioimmunoprecipitation with an antibody specific for the alteredtarget protein (or the target protein itself), binding of a ligandspecific for the altered target protein (or the target protein itself),or testing for a specific enzyme activity of the altered target protein(or the target protein itself).

In some embodiments, the isolated altered target molecules can befrozen, lyophilized, or immobilized and stored under appropriateconditions, e.g., which allow the altered target molecules to retainbiological activity.

The altered N-glycosylation form of the target molecule can be furtherprocessed in vivo (e.g., in the genetically engineered cell) or can beprocessed in vitro following isolation from the genetically engineeredcell or cell medium. The further processing can include modifications ofone or more N-glycan residues of the altered target molecule ormodifications to the altered target molecule other than to its N-glycanresidues. The additional processing of the altered target molecule caninclude the addition (covalent or non-covalent joining) of aheterologous moiety such as a polymer or a carrier. The furtherprocessing can also involve enzymatic or chemical treatment of thealtered target molecule. Enzymatic treatment can involve contacting thealtered target molecule with one or more of a glycosidase (e.g.,mannosidase or mannanase), a phosphodiesterase, a phospholipase, aglycosyltransferase, or a protease for a time sufficient to inducemodification of the altered target molecule. Enzymatic treatment canalso involve contacting the altered target molecule with an enzymecapable of removing one or more glucose residues from Man₅GlcNAc₂ suchas, but not limited to, a mannosidase or one or both of the alpha andbeta subunit of a glucosidase II. Chemical treatment can, for example,involve contacting the altered target molecule with an acid such ashydrofluoric acid for a time sufficient to induce modification of thealtered target molecule. Hydrofluoric acid treatment under certainconditions specifically removes the mannose residues that arephosphodiester-linked to glycans, while leaving the phosphate on theglycan. An altered target molecule can be further processed by additionor removal of a phosphate group from one or more N-glycans. For example,a altered target molecule can be contacted with a mannosyl kinase or amannosyl phosphatase.

In some embodiments, any of the altered target molecules describedherein, following isolation, can be attached to a heterologous moiety,e.g., using enzymatic or chemical means. A “heterologous moiety” refersto any constituent that is joined (e.g., covalently or non-covalently)to the altered target molecule, which constituent is different from aconstituent originally present on the altered target molecule.Heterologous moieties include, e.g., polymers, carriers, adjuvants,immunotoxins, or detectable (e.g., fluorescent, luminescent, orradioactive) moieties. In some embodiments, an additional N-glycan canbe added to the altered target molecule.

It is understood that a target molecule can be, but need not be,processed in a genetically engineered cell. For example, the disclosurealso features cell-free methods of producing a target molecule having analtered N-glycosylation form, which methods include the step ofcontacting a target molecule under N-glycosylation conditions with acell lysate prepared from a cell (e.g., a fungal cell (e.g., Yarrowialipolytica, Arxula adeninivorans, or any other related dimorphic yeastcells described herein), a plant cell, or an animal cell (e.g.,nematode, insect, plant, bird, reptile, or mammal (e.g., a mouse, rat,rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey,or human)) genetically engineered to have at least one modifiedN-glycosylation activity, wherein the contacting of the target moleculeto the cell lysate produces an altered N-glycosylation form of thetarget molecule.

By “N-glycosylation conditions” is meant that a mixture (e.g., of targetmolecule and cell lysate) is incubated under conditions that allow foraltered N-glycosylation (as described above).

Suitable methods for obtaining cell lysates that preserve the activityor integrity of one or more N-glycosylation activities in the lysate caninclude the use of appropriate buffers and/or inhibitors, includingnuclease, protease and phosphatase inhibitors that preserve or minimizechanges in N-glycosylation activities in the cell lysate. Suchinhibitors include, for example, chelators such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such asphenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain andthe like, and phosphatase inhibitors such as phosphate, sodium fluoride,vanadate and the like Inhibitors can be chosen such that they do notinterfere with or only minimally adversely affect the N-glycosylationactivity, or activities, of interest. Appropriate buffers and conditionsfor obtaining lysates containing enzymatic activities are described in,e.g., Ausubel et al. Current Protocols in Molecular Biology (Supplement47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press (1988); Harlow andLane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press(1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis andAshwood, eds. W.B. Saunders, Philadelphia, (1999).

A cell lysate can be further processed to eliminate or minimize thepresence of interfering substances, as appropriate. If desired, a celllysate can be fractionated by a variety of methods well known to thoseskilled in the art, including subcellular fractionation, andchromatographic techniques such as ion exchange, hydrophobic and reversephase, size exclusion, affinity, hydrophobic charge-inductionchromatography, and the like (see, e.g., Scopes, Protein Purification:Principles and Practice, third edition, Springer-Verlag, New York(1993); Burton and Harding, J. Chromatogr. A 814:71-81 (1998)).

In some embodiments, a cell lysate can be prepared in which wholecellular organelles remain intact and/or functional. For example, alysate can contain one or more of intact rough endoplasmic reticulum,intact smooth endoplasmic reticulum, or intact Golgi apparatus. Suitablemethods for preparing lysates containing intact cellular organelles andtesting for the functionality of the organelles are described in, e.g.,Moreau et al. (1991) J. Biol. Chem. 266(7):4329-4333; Moreau et al.(1991) J. Biol. Chem. 266(7):4322-4328; Rexach et al. (1991) J. CellBiol. 114(2):219-229; and Paulik et al. (1999) Arch. Biochem. Biophys.367(2):265-273; the disclosures of each of which are incorporated hereinby reference in their entirety.

The disclosure also provides methods of producing a target moleculehaving an altered N-glycosylation form that includes the step ofcontacting a target molecule under N-glycosylation conditions with oneor more isolated proteins having N-glycosylation activity, whereincontacting the target molecule with the one or more proteins havingN-glycosylation activity produces an altered N-glycosylation form of thetarget molecule and wherein the one or more proteins havingN-glycosylation activity are prepared from a cell (e.g., a fungal cell(e.g., Yarrowia lipolytica, Arxula adeninivorans, or any other relateddimorphic yeast cells described herein), a plant cell, or an animal cell(e.g., nematode, insect, plant, bird, reptile, or mammal (e.g., a mouse,rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale,monkey, or human)) genetically engineered to have at least one modifiedN-glycosylation activity.

One of more proteins having N-glycosylation activity can be purifiedusing standard techniques as described above. A target molecule can becontacted with one or more proteins in a suitable buffer for a timesufficient to induce modification of the target molecule as describedin, e.g., Lee and Park (2002) 30(6):716-720 and Fujita and Takegawa(2001) Biochem. Biophys. Res. Commun. 282(3):678-682, the disclosures ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the target molecule can be contacted with just oneprotein having N-glycosylation activity. In some embodiments, the targetmolecule can be contacted with more than one protein havingN-glycosylation activity. The target molecule can be contacted with morethan one protein at the same time or sequentially. Where the targetmolecule is contacted sequentially to more than one protein havingN-glycosylation activity, the target molecule can, but need not, bepurified after one or more steps. That is, a target molecule can becontacted with protein activity A, then purified before contacting themolecule to protein activity B, and so on.

It some embodiments of the cell free methods, it can be advantageous tolink the target molecule to a solid-phase support prior to contactingthe target molecule with one or more N-glycosylation activities. Suchlinkage can allow for easier purification following the N-glycosylationmodifications. Suitable solid-phase supports include, but are notlimited to, multi-well assay plates, particles (e.g., magnetic orencoded particles), a column, or a membrane.

Methods for detecting N-glycosylation (e.g., altered N-glycosylation) ofa target molecule include DNA sequencer-assisted (DSA),fluorophore-assisted carbohydrate electrophoresis (FACE) (as describedin the accompanying Examples) or surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)and. For example, an analysis can utilize DSA-FACE in which, forexample, glycoproteins are denatured followed by immobilization on,e.g., a membrane. The glycoproteins can then be reduced with a suitablereducing agent such as dithiothreitol (DTT) or β-mercaptoethanol. Thesulfhydryl groups of the proteins can be carboxylated using an acid suchas iodoacetic acid. Next, the N-glycans can be released from the proteinusing an enzyme such as N-glycosidase F. N-glycans, optionally, can bereconstituted and derivatized by reductive amination. The derivatizedN-glycans can then be concentrated. Instrumentation suitable forN-glycan analysis includes, e.g., the ABI PRISM® 377 DNA sequencer(Applied Biosystems). Data analysis can be performed using, e.g.,GENESCAN® 3.1 software (Applied Biosystems). Optionally, isolatedmannoproteins can be further treated with one or more enzymes to confirmtheir N-glycan status. Exemplary enzymes include, e.g., α-mannosidase orα-1,2 mannosidase, as described in the accompanying Examples. Additionalmethods of N-glycan analysis include, e.g., mass spectrometry (e.g.,MALDI-TOF-MS), high-pressure liquid chromatography (HPLC) on normalphase, reversed phase and ion exchange chromatography (e.g., with pulsedamperometric detection when glycans are not labeled and with UVabsorbance or fluorescence if glycans are appropriately labeled). Seealso Callewaert et al. (2001) Glycobiology 11(4):275-281 and Freire etal. (2006) Bioconjug. Chem. 17(2):559-564, the disclosures of each ofwhich are incorporated herein by reference in their entirety.

Disorders Treatable by Altered N-Glycosylation Molecules

The isolated, altered N-glycosylation molecules (e.g., the alteredN-glycosylation proteins or dolichol) described herein can be used totreat a variety of disorders, which disorders are treatable byadministration of one or more altered N-glycosylation molecules (e.g., aprotein having altered N-glycosylation). Examples of some specificmedical conditions that can be treated or prevented by administration ofan altered N-glycosylation molecule (e.g., an altered N-glycoprotein oran altered N-glycosylated dolichol) are reviewed in the followingsections.

(i) Metabolic Disorders

A metabolic disorder is one that affects the production of energy withinindividual human (or animal) cells. Most metabolic disorders aregenetic, though some can be “acquired” as a result of diet, toxins,infections, etc. Genetic metabolic disorders are also known as inbornerrors of metabolism. In general, the genetic metabolic disorders arecaused by genetic defects that result in missing or improperlyconstructed enzymes necessary for some step in the metabolic process ofthe cell. The largest classes of metabolic disorders are disorders ofcarbohydrate metabolism, disorders of amino acid metabolism, disordersof organic acid metabolism (organic acidurias), disorders of fatty acidoxidation and mitochondrial metabolism, disorders of porphyrinmetabolism,

disorders of purine or pyrimidine metabolism, disorders of steroidmetabolism disorders of mitochondrial function, disorders of peroxisomalfunction, and lysosomal storage disorders (LSDs).

Examples of metabolic disorders that can be treated through theadministration of one or more altered N-glycosylation molecules (orpharmaceutical compositions of the same) described herein can include,e.g., hereditary hemochromatosis, oculocutaneous albinism, protein Cdeficiency, type I hereditary angioedema, congenital sucrase-isomaltasedeficiency, Crigler-Najjar type II, Laron syndrome, hereditaryMyeloperoxidase, primary hypothyroidism, congenital long QT syndrome,tyroxine binding globulin deficiency, familial hypercholesterolemia,familial chylomicronemia, abeta-lipoproteinema, low plasma lipoprotein Alevels, hereditary emphysema with liver injury, congenitalhypothyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,alpha-1 antichymotrypsin deficiency, nephrogenic diabetes insipidus,neurohypophyseal diabetes insipidus, adenosine deaminase deficiency,Pelizaeus Merzbacher disease, von Willebrand disease type HA, combinedfactors V and VIII deficiency, spondylo-epiphyseal dysplasia tarda,choroideremia, I cell disease, Batten disease, ataxia telangiectasias,ADPKD-autosomal dominant polycystic kidney disease, microvillusinclusion disease, tuberous sclerosis, oculocerebro-renal syndrome ofLowe, amyotrophic lateral sclerosis, myelodysplastic syndrome, Barelymphocyte syndrome, Tangier disease, familial intrahepatic cholestasis,X-linked adreno-leukodystrophy, Scott syndrome, Hermansky-Pudlaksyndrome types 1 and 2, Zellweger syndrome, rhizomelic chondrodysplasiapuncta, autosomal recessive primary hyperoxaluria, Mohr Tranebjaergsyndrome, spinal and bullar muscular atrophy, primary ciliary diskenesia(Kartagener's syndrome), giantism and acromegaly, galactorrhea,Addison's disease, adrenal virilism, Cushing's syndrome, ketoacidosis,primary or secondary aldosteronism, Miller Dieker syndrome,lissencephaly, motor neuron disease, Usher's syndrome, Wiskott-Aldrichsyndrome, Optiz syndrome, Huntington's disease, hereditary pancreatitis,anti-phospholipid syndrome, overlap connective tissue disease, Sjögren'ssyndrome, stiff-man syndrome, Brugada syndrome, congenital nephriticsyndrome of the Finnish type, Dubin-Johnson syndrome, X-linkedhypophosphosphatemia, Pendred syndrome, persistent hyperinsulinemichypoglycemia of infancy, hereditary spherocytosis, aceruloplasminemia,infantile neuronal ceroid lipofuscinosis, pseudoachondroplasia andmultiple epiphyseal, Stargardt-like macular dystrophy, X-linkedCharcot-Marie-Tooth disease, autosomal dominant retinitis pigmentosa,Wolcott-Rallison syndrome, Cushing's disease, limb-girdle musculardystrophy, mucoploy-saccharidosis type IV, hereditary familialamyloidosis of Finish, Anderson disease, sarcoma, chronic myelomonocyticleukemia, cardiomyopathy, faciogenital dysplasia, Torsion disease,Huntington and spinocerebellar ataxias, hereditary hyperhomosyteinemia,polyneuropathy, lower motor neuron disease, pigmented retinitis,seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud'sphenomenon, Wegner's granulomatosis, preoteinuria, CDG-Ia, CDG-Ib,CDG-Ic, CDG-Id, CDG-Ie, CDG-If, CDG-IIa, CDG-IIb, CDG-IIc, CDG-IId,Ehlers-Danlos syndrome, multiple exostoses, Griscelli syndrome (type 1or type 2), or X-linked non-specific mental retardation. In addition,metabolic disorders can also include lysosomal storage disorders suchas, but not limited to, Fabry disease, Farber disease, Gaucher disease,GM₁-gangliosidosis, Tay-Sachs disease, Sandhoff disease, GM₂ activatordisease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pickdisease (types A, B, and C), Hurler disease, Scheie disease, Hunterdisease, Sanfilippo disease, Morquio disease, Maroteaux-Lamy disease,hyaluronidase deficiency, aspartylglucosaminuria, fucosidosis,mannosidosis, Schindler disease, sialidosis type 1, Pompe disease,Pycnodysostosis, ceroid lipofuscinosis, cholesterol ester storagedisease, Wolman disease, Multiple sulfatase deficiency,galactosialidosis, mucolipidosis (types II, III, and IV), cystinosis,sialic acid storage disorder, chylomicron retention disease withMarinesco-Sjögren syndrome, Hermansky-Pudlak syndrome, Chediak-Higashisyndrome, Danon disease, or Geleophysic dysplasia.

Symptoms of a metabolic disorder are numerous and diverse and caninclude one or more of, e.g., anemia, fatigue, bruising easily, lowblood platelets, liver enlargement, spleen enlargement, skeletalweakening, lung impairment, infections (e.g., chest infections orpneumonias), kidney impairment, progressive brain damage, seizures,extra thick meconium, coughing, wheezing, excess saliva or mucousproduction, shortness of breath, abdominal pain, occluded bowel or gut,fertility problems, polyps in the nose, clubbing of the finger/toe nailsand skin, pain in the hands or feet, angiokeratoma, decreasedperspiration, corneal and lenticular opacities, cataracts, mitral valveprolapse and/or regurgitation, cardiomegaly, temperature intolerance,difficulty walking, difficulty swallowing, progressive vision loss,progressive hearing loss, hypotonia, macroglossia, areflexia, lower backpain, sleep apnea, orthopnea, somnolence, lordosis, or scoliosis. It isunderstood that due to the diverse nature of the defective or absentproteins and the resulting disease phenotypes (e.g., symptomaticpresentation of a metabolic disorder), a given disorder will generallypresent only symptoms characteristic to that particular disorder. Forexample, a patient with Fabry disease can present a particular subset ofthe above-mentioned symptoms such as, but not limited to, temperatureintolerance, corneal whirling, pain, skin rashes, nausea, or dirarrhea.A patient with Gaucher syndrome can present with splenomegaly,cirrhosis, convulsions, hypertonia, apnea, osteoporosis, or skindiscoloration.

In addition to the administration of one or more altered N-glycosylationmolecules described herein, a metabolic disorder can also be treated byproper nutrition and vitamins (e.g., cofactor therapy), physicaltherapy, and pain medications.

Depending on the specific nature of a given metabolic disorder, apatient can present these symptoms at any age. In many cases, symptomscan present in childhood or in early adulthood. For example, symptoms ofFabry disease can present at an early age, e.g., at 10 or 11 years ofage.

As used herein, a subject “at risk of developing a metabolic disorder”(such as one described herein) is a subject that has a predisposition todevelop a disorder, i.e., a genetic predisposition to develop metabolicdisorder as a result of a mutation in a enzyme such asalpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase,beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase,arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase,aspartylglucosaminidase, iduronate-2-sulfatase,alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase,hyaluronidase, alpha-L-mannosidase, alpha-neuromimidase,phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase,thioesterase, cathepsin K, or lipoprotein lipase. Clearly, subjects “atrisk of developing a metabolic disorder” are not all the subjects withina species of interest.

A subject “suspected of having a disorder” is one having one or moresymptoms of a disorder (e.g., a metabolic disorder or any other disorderdescribed herein) such as any of those described herein.

(ii) Cancer

Cancer is a class of diseases or disorders characterized by uncontrolleddivision of cells and the ability of these to spread, either by directgrowth into adjacent tissue through invasion, or by implantation intodistant sites by metastasis (where cancer cells are transported throughthe bloodstream or lymphatic system). Cancer can affect people at allages, but risk tends to increase with age. Types of cancers can include,e.g., lung cancer, breast cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer,neural tissue cancer, melanoma, thyroid cancer, ovarian cancer,testicular cancer, prostate cancer, cervical cancer, vaginal cancer, orbladder cancer.

As used herein, a subject “at risk of developing a cancer” is a subjectthat has a predisposition to develop a cancer, i.e., a geneticpredisposition to develop cancer such as a mutation in a tumorsuppressor gene (e.g., mutation in BRCA1, p53, RB, or APC) or has beenexposed to conditions that can result in cancer. Thus, a subject canalso be one “at risk of developing a cancer” when the subject has beenexposed to mutagenic or carcinogenic levels of certain compounds (e.g.,carcinogenic compounds in cigarette smoke such as Acrolein, Arsenic,Benzene, Benz{a}anthracene, Benzo{a}pyrene, Polonium-210 (Radon),Urethane, or Vinyl Chloride). Moreover, the subject can be “at risk ofdeveloping a cancer” when the subject has been exposed to, e.g., largedoses of ultraviolet light or X-irradiation, or exposed (e.g., infected)to a tumor-causing/associated virus such as papillomavirus, Epstein-Barrvirus, hepatitis B virus, or human T-cell leukemia-lymphoma virus. Fromthe above it will be clear that subjects “at risk of developing acancer” are not all the subjects within a species of interest.

A subject “suspected of having a cancer” is one having one or moresymptoms of a cancer. Symptoms of cancer are well-known to those ofskill in the art and include, without limitation, breast lumps, nipplechanges, breast cysts, breast pain, weight loss, weakness, excessivefatigue, difficulty eating, loss of appetite, chronic cough, worseningbreathlessness, coughing up blood, blood in the urine, blood in stool,nausea, vomiting, liver metastases, lung metastases, bone metastases,abdominal fullness, bloating, fluid in peritoneal cavity, vaginalbleeding, constipation, abdominal distension, perforation of colon,acute peritonitis (infection, fever, pain), pain, vomiting blood, heavysweating, fever, high blood pressure, anemia, diarrhea, jaundice,dizziness, chills, muscle spasms, colon metastases, lung metastases,bladder metastases, liver metastases, bone metastases, kidneymetastases, and pancreas metastases, difficulty swallowing, and thelike.

In addition to the administration of one or more altered N-glycosylationmolecules described herein, a cancer can also be treated bychemotherapeutic agents, ionizing radiation, immunotherapy agents, orhyperthermotherapy agents. Chemotherapeutic agents include, e.g.,cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen,taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, andmethotrexate.

(iii) Inflammatory Disorders

An “inflammatory disorder,” as used herein, refers to a process in whichone or more substances (e.g., substances not naturally occurring in thesubject), via the action of white blood cells (e.g., B cells, T cells,macrophages, monocytes, or dendritic cells) inappropriately trigger apathological response, e.g., a pathological immune response.Accordingly, such cells involved in the inflammatory response arereferred to as “inflammatory cells.” The inappropriately triggeredinflammatory response can be one where no foreign substance (e.g., anantigen, a virus, a bacterium, a fungus) is present in or on thesubject. The inappropriately triggered response can be one where aself-component (e.g., a self-antigen) is targeted (e.g., an autoimmunedisorder such as multiple sclerosis) by the inflammatory cells. Theinappropriately triggered response can also be a response that isinappropriate in magnitude or duration, e.g., anaphylaxis. Thus, theinappropriately targeted response can be due to the presence of amicrobial infection (e.g., viral, bacterial, or fungal). Types ofinflammatory disorders (e.g., autoimmune disease) can include, but arenot limited to, osteoarthritis, rheumatoid arthritis (RA),spondyloarthropathies, POEMS syndrome, Crohn's disease, multicentricCastleman's disease, systemic lupus erythematosus (SLE), multiplesclerosis (MS), muscular dystrophy (MD), insulin-dependent diabetesmellitus (IDDM), dermatomyositis, polymyositis, inflammatoryneuropathies such as Guillain Barre syndrome, vasculitis such asWegener's granulomatosus, polyarteritis nodosa, polymyalgia rheumatica,temporal arteritis, Sjogren's syndrome, Bechet's disease, Churg-Strausssyndrome, or Takayasu's arteritis. Also included in inflammatorydisorders are certain types of allergies such as rhinitis, sinusitis,urticaria, hives, angioedema, atopic dermatitis, food allergies (e.g., anut allergy), drug allergies (e.g., penicillin), insect allergies (e.g.,allergy to a bee sting), or mastocytosis. Inflammatory disorders canalso include ulcerative colitis and asthma.

A subject “at risk of developing an inflammatory disorder” refers to asubject with a family history of one or more inflammatory disorders(e.g., a genetic predisposition to one or more inflammatory disorders)or one exposed to one or more inflammation-inducing conditions.

For example, a subject can have been exposed to a viral or bacterialsuperantigen such as, but not limited to, staphylococcal enterotoxins(SEs), a streptococcus pyogenes exotoxin (SPE), a staphylococcus aureustoxic shock-syndrome toxin (TSST-1), a streptococcal mitogenic exotoxin(SME) and a streptococcal superantigen (SSA). From the above it will beclear that subjects “at risk of developing an inflammatory disorder” arenot all the subjects within a species of interest.

A subject “suspected of having an inflammatory disorder” is one whopresents with one or more symptoms of an inflammatory disorder. Symptomsof inflammatory disorders are well known in the art and include, but arenot limited to, redness, swelling (e.g., swollen joints), joints thatare warm to the touch, joint pain, stiffness, loss of joint function,fever, chills, fatigue, loss of energy, headaches, loss of appetite,muscle stiffness, insomnia, itchiness, stuffy nose, sneezing, coughing,one or more neurologic symptoms such as dizziness, seizures, or pain.

In addition to the administration of one or more altered N-glycosylationmolecules described herein, an inflammatory disorder can also be treatedby non-steroidal anti-inflammatory drug (NSAID), a disease-modifyinganti-rheumatic drug (DMARD), a biological response modifier, or acorticosteroid. Biological response modifiers include, e.g., an anti-TNFagent (e.g., a soluble TNF receptor or an antibody specific for TNF suchas adulimumab, infliximab, or etanercept).

Methods suitable for treating (e.g., preventing or ameliorating one ormore symptoms of) any of the disorders described herein using any of thealtered N-glycosylation molecules (or pharmaceutical compositionsthereof) are set forth in the following section.

Pharmaceutical Compositions and Methods of Treatment

An altered N-glycosylation molecule (e.g., an altered N-glycosylationform of a target molecule such as a target protein) can be incorporatedinto a pharmaceutical composition containing a therapeutically effectiveamount of the molecule and one or more adjuvants, excipients, carriers,and/or diluents. Acceptable diluents, carriers and excipients typicallydo not adversely affect a recipient's homeostasis (e.g., electrolytebalance). Acceptable carriers include biocompatible, inert orbioabsorbable salts, buffering agents, oligo- or polysaccharides,polymers, viscosity-improving agents, preservatives and the like. Oneexemplary carrier is physiologic saline (0.15 M NaCl, pH 7.0 to 7.4).Another exemplary carrier is 50 mM sodium phosphate, 100 mM sodiumchloride. Further details on techniques for formulation andadministration of pharmaceutical compositions can be found in, e.g.,Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).Supplementary active compounds can also be incorporated into thecompositions.

Administration of a pharmaceutical composition containing an alteredN-glycosylation molecule can be systemic or local. Pharmaceuticalcompositions can be formulated such that they are suitable forparenteral and/or non-parenteral administration. Specific administrationmodalities include subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, intrathecal, oral, rectal, buccal,topical, nasal, ophthalmic, intra-articular, intra-arterial,sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterineadministration.

Administration can be by periodic injections of a bolus of thepharmaceutical composition or can be uninterrupted or continuous byintravenous or intraperitoneal administration from a reservoir which isexternal (e.g., an IV bag) or internal (e.g., a bioerodable implant, abioartificial organ, or a colony of implanted altered N-glycosylationmolecule production cells). See, e.g., U.S. Pat. Nos. 4,407,957,5,798,113, and 5,800,828, each incorporated herein by reference in theirentirety. Administration of a pharmaceutical composition can be achievedusing suitable delivery means such as: a pump (see, e.g., Annals ofPharmacotherapy, 27:912 (1993); Cancer, 41:1270 (1993); Cancer Research,44:1698 (1984), incorporated herein by reference in its entirety);microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and5,084,350, herein incorporated by reference in its entirety); continuousrelease polymer implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666,incorporated herein by reference in its entirety); macroencapsulation(see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733and published PCT patent applications WO92/19195, WO 95/05452, thedisclosures of each of which are incorporated herein by reference intheir entirety); injection, either subcutaneously, intravenously,intra-arterially, intramuscularly, or to other suitable site; or oraladministration, in capsule, liquid, tablet, pill, or prolonged releaseformulation.

Examples of parenteral delivery systems include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, pumpdelivery, encapsulated cell delivery, liposomal delivery,needle-delivered injection, needle-less injection, nebulizer,aerosolizer, electroporation, and transdermal patch.

Formulations suitable for parenteral administration conveniently containa sterile aqueous preparation of the altered N-glycosylation molecule,which preferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Formulations can be presented inunit-dose or multi-dose form.

Formulations suitable for oral administration can be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the altered N-glycosylationmolecule; or a suspension in an aqueous liquor or a non-aqueous liquid,such as a syrup, an elixir, an emulsion, or a draught.

An altered N-glycosylation molecule (e.g., an altered N-glycosylationform of a target molecule such as a target protein) suitable for topicaladministration can be administered to a mammal (e.g., a human patient)as, e.g., a cream, a spray, a foam, a gel, an ointment, a salve, or adry rub. A dry rub can be rehydrated at the site of administration. Analtered N-glycosylation molecule can also be infused directly into(e.g., soaked into and dried) a bandage, gauze, or patch, which can thenbe applied topically. Altered N-glycosylation molecules can also bemaintained in a semi-liquid, gelled, or fully-liquid state in a bandage,gauze, or patch for topical administration (see, e.g., U.S. Pat. No.4,307,717, the content of which is incorporated herein by reference inits entirety).

Therapeutically effective amounts of a pharmaceutical composition can beadministered to a subject in need thereof in a dosage regimenascertainable by one of skill in the art. For example, a composition canbe administered to the subject, e.g., systemically at a dosage from 0.01μg/kg to 10,000 μg/kg body weight of the subject, per dose. In anotherexample, the dosage is from 1 μg/kg to 100 μg/kg body weight of thesubject, per dose. In another example, the dosage is from 1 μg/kg to 30μg/kg body weight of the subject, per dose, e.g., from 3 μg/kg to 10μg/kg body weight of the subject, per dose.

In order to optimize therapeutic efficacy, an altered N-glycosylationmolecule can be first administered at different dosing regimens. Theunit dose and regimen depend on factors that include, e.g., the speciesof mammal, its immune status, the body weight of the mammal. Typically,levels of an altered N-glycosylation molecule in a tissue can bemonitored using appropriate screening assays as part of a clinicaltesting procedure, e.g., to determine the efficacy of a given treatmentregimen.

The frequency of dosing for an altered N-glycosylation molecule iswithin the skills and clinical judgement of medical practitioners (e.g.,doctors or nurses). Typically, the administration regime is establishedby clinical trials which may establish optimal administrationparameters. However, the practitioner may vary such administrationregimes according to the subject's age, health, weight, sex and medicalstatus. The frequency of dosing can be varied depending on whether thetreatment is prophylactic or therapeutic.

Toxicity and therapeutic efficacy of such altered N-glycosylationmolecules (e.g., an altered N-glycosylation form of target moleculessuch as target proteins) or pharmaceutical compositions thereof can bedetermined by known pharmaceutical procedures in, for example, cellcultures or experimental animals. These procedures can be used, e.g.,for determining the LD50 (the dose lethal to 50% of the population) andthe ED50 (the dose therapeutically effective in 50% of the population).The dose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Pharmaceuticalcompositions that exhibit high therapeutic indices are preferred. Whilepharmaceutical compositions that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to normal cells (e.g., non-target cells) and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in appropriate subjects(e.g., human patients). The dosage of such pharmaceutical compositionslies generally within a range of circulating concentrations that includethe ED50 with little or no toxicity. The dosage may vary within thisrange depending upon the dosage form employed and the route ofadministration utilized. For a pharmaceutical composition used asdescribed herein (e.g., for treating a metabolic disorder in a subject),the therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the pharmaceutical composition which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

As defined herein, a “therapeutically effective amount” of an alteredN-glycosylation molecule is an amount of the molecule that is capable ofproducing a medically desirable result (e.g., amelioration of one ormore symptoms of a metabolic disorder or decreased proliferation ofcancer cells) in a treated subject. A therapeutically effective amountof an altered N-glycosylation molecule (i.e., an effective dosage)includes milligram or microgram amounts of the compound per kilogram ofsubject or sample weight (e.g., about 1 microgram per kilogram to about500 milligrams per kilogram, about 100 micrograms per kilogram to about5 milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram).

The subject can be any mammal, e.g., a human (e.g., a human patient) ora non-human primate (e.g., chimpanzee, baboon, or monkey), a mouse, arat, a rabbit, a guinea pig, a gerbil, a hamster, a horse, a type oflivestock (e.g., cow, pig, sheep, or goat), a dog, a cat, or a whale.

An altered N-glycosylation molecule or pharmaceutical compositionthereof described herein can be administered to a subject as acombination therapy with another treatment, e.g., a treatment for ametabolic disorder (e.g., a lysosomal storage disorder). For example,the combination therapy can include administering to the subject (e.g.,a human patient) one or more additional agents that provide atherapeutic benefit to the subject who has, or is at risk of developing,(or suspected of having) a metabolic disorder (e.g., a lysosomal storagedisorder). Thus, the compound or pharmaceutical composition and the oneor more additional agents are administered at the same time.Alternatively, the altered N-glycosylation molecule (e.g., protein ordolichol) can be administered first in time and the one or moreadditional agents administered second in time. The one or moreadditional agents can be administered first in time and the alteredN-glycosylation molecule (e.g., protein or dolichol) administered secondin time. The altered N-glycosylation molecule can replace or augment apreviously or currently administered therapy. For example, upon treatingwith an altered N-glycosylation molecule of the invention,administration of the one or more additional agents can cease ordiminish, e.g., be administered at lower levels. Administration of theprevious therapy can also be maintained. In some instances, a previoustherapy can be maintained until the level of the altered N-glycosylationmolecule (e.g., the dosage or schedule) reaches a level sufficient toprovide a therapeutic effect. The two therapies can be administered incombination.

It will be appreciated that in instances where a previous therapy isparticularly toxic (e.g., a treatment for a metabolic disorder withsignificant side-effect profiles), administration of the alteredN-glycosylation molecule (e.g., protein or dolichol) can be used tooffset and/or lessen the amount of the previously therapy to a levelsufficient to give the same or improved therapeutic benefit, but withoutthe toxicity.

In some instances, when the subject is administered an alteredN-glycosylation molecule (e.g., protein, dolichol, or a dolichol-linkedlipid) or pharmaceutical composition of the invention the first therapyis halted. The subject can be monitored for a first pre-selected result,e.g., an improvement in one or more symptoms of a metabolic disordersuch as any of those described herein (e.g., see above). In some cases,where the first pre-selected result is observed, treatment with thealtered N-glycosylation molecule (e.g., an altered N-glycosylationprotein or an altered N-glycosylation dolichol) is decreased or halted.The subject can then be monitored for a second pre-selected result aftertreatment with the altered N-glycosylation molecule (e.g., protein ordolichol) is halted, e.g., a worsening of a symptom of a metabolicdisorder. When the second pre-selected result is observed,administration of the altered N-glycosylation molecule (e.g., protein ordolichol) to the subject can be reinstated or increased, oradministration of the first therapy is reinstated, or the subject isadministered both an altered N-glycosylation molecule (e.g., protein,dolichol, or a dolichol-linked lipid) and first therapy, or an increasedamount of the altered N-glycosylation molecule (e.g., protein ordolichol) and the first therapeutic regimen.

The altered N-glycosylation molecule (e.g., protein or dolichol) canalso be administered with a treatment for one or more symptoms of adisease (e.g., a metabolic disorder). For example, the alteredN-glycosylation molecule (e.g., protein, dolichol, or a dolichol-linkedlipid) can be co-administered (e.g., at the same time or by anycombination regimen described above) with, e.g., a pain medication.

It is understood that in some embodiments, an altered N-glycosylationmolecule is one in which the altered glycosylation increases the abilityof the molecule to produce a medically relevant product. For example, analtered N-glycosylation molecule can be an enzyme capable of producing atherapeutic product (e.g., a small molecule or therapeutic peptide),which enzyme's activity is increased or optimized by glycosylation. Suchproducts and methods of using the products are within the scope of thepresent disclosure.

Any of the pharmaceutical compositions described herein can be includedin a container, pack, or dispenser together with instructions foradministration.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Plasmids, Primers and Strains

Table 1 contains a list of all of the plasmids used in the constructionof vectors (e.g., expression vectors) and deletion cassettes used in theexperiments described herein. The MTLY60 strain of Yarrowia lipolyticawas used in the experiments.

Table 2 contains a list of primers (the names of the primers) and theutility of the primers used in the following examples.

TABLE 1 Plasmids: JMP62 pYLTsA pYLHmL pYLHmA JMP113 JMP114 pRRQ2 JME 507JME 509 JME 461 KS-LPR- URA3 KS-LPR-LEU2 Cre ARS68 LEU2

TABLE 2 Primers: Name: Use: TCGCTATCACGTCTCTAGC Yloch1 prom fwAmplification YlOCH1 (SEQ ID NO: 18) Amplification YlOCH1 P fragmentTCTCTGTATACTTGTATGT Yloch1 ter rev Amplification YlOCH1ACTG (SEQ ID NO: 19) Amplification YlOCH1 T fragment CTAGGGATAACAGGGTAAYlOCH1 Pfrag rev Amplification P fragment incl  TGGTGTGACGAAGTATCGI-SceI site AG (SEQ ID NO: 20) CATTACCCTGTTATCCCTA YlOCH1 Tfrag fwAmplification T fragment incl  GCGAGATCATGGACTGG I-SceI site(SEQ ID NO: 21) GACGCGGCCGCATGAGCT YlMNS1 ORF + TerAmplification of YlMNS1 P frag. TCAACATTCCCAAAAC (Pfrag) S (ORF +terminator) (SEQ ID NO: 22) CTAGGGATAACAGGGTAA YlMNS1 ORF + TerAmplification of YlMNS1 P frag. TACAAAATTCAGAAATAA (Pfrag) AS (ORF +termin.) + I-SceI AAATACTTTACAG  (SEQ ID NO: 23) CATTACCCTGTTATCCCTAYlMNS1 Tfrag S Amplification of YlMNS1 T frag. AGTAACATGAGTGCTATG(downstream terminator.) + I-SceI AG (SEQ ID NO: 24) CGCTTAATTAAATGCATGGYlMNS1 Tfrag AS Amplification of YlMNS1 T frag. AGGTATTGCTG (downstream terminator.) (SEQ ID NO: 25) GGTGCTTCGACTATCAGTTScMNS1 mut 269-273  ScMNS1 mutation primer to shift toTCGGAGGATTGGGTGATTC S mam. Golgi type mannase => proofTTTTTATG (SEQ ID NO: 26) of concept in Sc CATAAAAAGAATCACCCAScMNS1 mut 269-273 YlMNS1 mutation primer to shift toATCCTCCGAAACTGATAGT AS mam. Golgi type mannase => proof CGAAGCACC of concept in Sc (SEQ ID NO: 27) TGAGCGGCCGCTTTTCTAC YlMNN9 P fwYlMNN9 KO primer TTCAGAGCTGGAG  (SEQ ID NO: 28) GGCTTAATTAATTGGTAGTYlMNN9 T rv YlMNN9 KO primer GATATAATGTAACGC  (SEQ ID NO: 29)TAGGGATAACAGGGTAAT YlMNN9 P rv YlMNN9 KO primer CACGACACATACTCATTCAAG (SEQ ID NO: 30) ATTACCCTGTTATCCCTAG YlMNN9 T fw YlMNN9 KO primerAAGGAGATGTAGCGTAAG (SEQ ID NO: 31) TGATAAATAGCTTAGATAC LIP2 rvReverse primer used for sequencing CACAG (SEQ ID NO: 32)ACATACAACCACACACATC 5′ hp4d Forward primer used for sequencing(SEQ ID NO: 33) GGCGGATCCATGGTGCTGC YlMNN4 BamHI fwForward primer for amplification of ACCCGTTTC (SEQ ID NO: 34) YlMNN4GGCCCTAGGCTACTCAAAC YlMNN4 AvrII rv Reverse primer for amplification ofTCCTCGCGAATC  YlMNN4 (SEQ ID NO: 35) GGTCTCGCCAGCGCGCCCA HAC1FW06-003Forward primer region around CCCTCTTC (SEQ ID NO: 36) HAC1 splice siteCTAGATCAGCAATAAAGT HAC1Rv06-001 Reverse primer region around HAC1CGTGCTGGGC  splice site (SEQ ID NO: 37) GGATCCATGTCTATCAAGC HAC1Fw06-002Amplification of HAC1 gene GAGAAGAG TCC  includes start codon and BamHI(SEQ ID NO: 38) restriction site CCTAGGCTAGATCAGCAAT HAC1RV06-006Amplification of HAC1 gene AAAGTCGTGCTGGGC includes stop codon and AvrII (SEQ ID NO: 39) restriction site

Example 2 Yarrowia lipolytica OCH1 and MNN9 Disruption

A strategy to knock out both OCH1 (GenBank® Accession No: AJ563920) andMNN9 (GenBank® Accession No: AF441127) genes in Yarrowia lipolytica wasset up as described in Fickers et al. ((2003) J Microbiol Methods.55(3):727-37) for the LIP2 gene. The gene construction strategy that wasfollowed for the OCH1 gene is depicted in FIGS. 5A-5E.

The OCH1 KO fragment was isolated from the plasmid YlOCH1 PUT TOPO byrestriction digest and by PCR and was transformed to Yarrowia lipolyticastrain MTLY60. 20 uracil prototrophic strains were obtained and screenedby PCR on genomoic DNA (gDNA) using primers Yloch1 prom fw (SEQ IDNO:18) and Yloch1 ter rev (SEQ ID NO:19) to analyse the genomicintegration of the plasmid. A fragment of the correct size (i.e., 2618bp vs. 1894 bp in the wild type) was amplified in 2 of the 20 clonestested. Several clones contained a random integrated copy of theconstruct and therefore both fragments were amplified.

To remove the URA3 gene, the two positive clones were transformed withthe episomal plasmid pRRQ2 that contains an expression cassette for theCre recombinase. Removal of the URA3 gene was screened for by PCR ongDNA using primers Yloch1 prom fw and Yloch1 ter rev (see above). The2328 bp fragment (incl. URA3) was absent from, and a 1075 bp (excl.URA3) fragment of 1075 bp was present in, the positive clones.

A Southern blot analysis was performed on the 2 positive clones to checkwhether aberrant DNA integration had occurred. Genomic DNA (gDNA) wasdouble digested with EcoRV/HindIII, subjected to agarose-gelelectrophoresis, and transferred to nitrocellulose membrane. Themembrane was probed with a 500 bp SpeI/1-SceI fragment from plasmidYlOCH1 PT TOPO. A fragment of 1456 bp was present in Δoch1 PUT, whereasa fragment of 2066 bp in Δoch1 PT and a fragment of 2893 bp in the wildtype strain was present.

A construction strategy to inactivate MNN9 was set up and is depicted inFIGS. 6A and 6B.

The disruption fragment was cut out of plasmid YlMNN9PUT TOPO by aNotI/PacI double digest and transformed to MTLY60 and Δoch1 PT clone 9.Several URA3 positive clones were obtained for both strains and theywere screened for correct integration of the construct by PCR on gDNAafter single clones were isolated. A fragment of 2349 bp was amplifiedin the disruptant strains, whereas in the non-transformants, a fragmentof 2056 bp was amplified using primers YlMNN9 P fw and YlMNN9 T rv.(Table 2).

To analyze the N-glycan structures that were synthesized by the mutantstrains, DSA-FACE was performed on glycans derived from mannoproteins(FIG. 7). The wild-type (MTLY60) strain has as main core type glycanstructures mainly Man₈GlcNAc₂ (structural formula I; FIG. 4A) and asubstantial amount of Man₉GlcNAc₂ (structural formula II; FIG. 4A) thelatter most probably containing an additional mannose as a result ofOch1p activity. Furthermore, some larger structures can be seen. TheΔoch1 strain has mainly Man₈GlcNAc₂ (structure formula I) and a smallportion of Man₉GlcNAc₂ (structural formula II; FIG. 4A), both of whichare sensitive to α-1,2-mannosidase treatment (indicated Δoch1 α-1,2-man)resulting in trimming to Man₅GlcNAc₂ (structural formula IV; FIG. 4A).The Δmnn9 strain accumulates more Man₉GlcNAc₂ (structural formula II;FIG. 4A) than the Δoch1 strain, which indicates that Mnn9p is involvedin the elongation of the glycan structure subsequent to Och1p activity.The double mutant Δoch1 Δmnn9 displays a glycosylation phenotype thatresembles the one from the Δoch1 strain.

Example 3 Mutagenesis of MNS1

MNS1 (ER α-1,2-mannosidase) is involved in the trimming of theMan₉GlcNAc₂ to Man₈GlcNAc₂ and has a strict substrate specificity in thesense that it is only able to trim the α-1,2-mannose that is linked tothe α-1,3-mannose of the central arm (FIG. 2). To determine where theMNS1 gene could be mutagenized in order to shift its substratespecificity towards a Golgi type α-1,2-mannosidase, the primarysequences of several ER type mannosidases were compared with Golgi typemannosidases. One region that is different between the two classes wasidentified. In addition, an oligosaccharide that was crystallised in thecatalytic site of the Golgi type mannosidase into the yeast MNS1 wasalso analyzed to identify possible interactions between sugar andprotein. Surprisingly, the same sites were identified using bothmethods. The MNS1 gene from Saccharomyces cerevisiae (GenBank® AccessionNo: Z49631, sgd: YJR131W) was mutated in order to change its substratespecificity. Three mutated versions were made: two with one mutation(R273L and R273G) and one with 3 mutations (R269S/S272G/R273L) in thesame region:

-   A) R273L (arginine 273 to leucine)-   B) R273G (arginine 273 to glycine)-   C) R269S/S272G/R273L (arginine 269 to serine/serine 272 to    glycine/arginine 273 to leucine).

All mutations were made using the Quick Change (Stratagene) mutagenesiskit. Constructs were made to express the 3 different mutant genes undercontrol of the strong constitutive TPI1 promoter. OligonucleotidesCGACTATCCGGTTCGGATCATTGGGTGATTCTTTTTATGAG (SEQ ID NO:40) andCTCATAAAAAGAATCACCCAATGATCCGAACCGGATAGTCG (SEQ ID NO:41) were used togenerate mutant R273L, and oligonucleotidesCGACTATCCGGTTCGGATCAGGTGGTGATTCTTTTTATGAG (SEQ ID NO:42) andCTCATAAAAAGAATCACCACCTGATCCGAACCGGATAGTCG (SEQ ID NO:43) were used toobtain mutant R273G using the wild type gene as a template.Oligonucleotides GGTGCTTCGACTATCAGTTTCGGAGGATTGGGTGATTCTTTTTATG (SEQ IDNO:44) and CATAAAAAGAATCACCCAATCCTCCGAAACTGATAGTCGAAGCACC (SEQ ID NO:45)were used to obtain mutant R269S/S272G/R273L using mutant R273L astemplate DNA. Via PCR reaction using oligonucleotidesCCCGATATCGGATCCATGAAGAACTCTGTCGGTATTTC (SEQ ID NO:46) andGGGAAGCTTAACGCGGTTCCAGCGGGTCCGGATACGGCACCGGCGCACCCAACGACCAACCTGTGGTCAG(SEQ ID NO:47) the coding sequence of an E-tag was added at the 3′ endof the mutant and the wild type MNSlopen reading frames to allow proteindetection after expression. An overview of the construction strategy ispresented in FIGS. 8A-8E.

The three constructs, as well as the non-mutated gene (as a negativecontrol), were transformed to S. cerevisiae strain XW27 (MATα leu2 ura3trp1 his3 ade2 lys2 och1::LEU2 mnn1::URA3 nm n6::ADE2) using TRP1 as aselection marker after digestion of the plasmids with XbaI to direct theconstruct to the TRP1 locus in the S. cerevisiae genome. The latterstrain is able to synthesize uniform Man₈GlcNAc₂ (on its glycoproteins.If the mutated enzyme is active this Man₈GlcNAc₂ (structural formula I;FIG. 4A) should be trimmed to Man₅GlcNAc₂ (structural formula IV; FIG.4A), Man₆GlcNAc₂ (structural formula V; FIG. 4A) and/or Man₇GlcNAc₂(structural formula VI; FIG. 4A).

Tryptophan prototrophic strains were isolated, grown in liquid SDC-trpmedium and mannoproteins were prepared. N-glycans derived frommannoproteins were analysed via DSA-FACE. As can be appreciated fromFIG. 9, a small amount of Man₈GlcNAc₂ (structural formula I; FIG. 4A)from the strains that contain the R273G and R269S/S272G/R273L mutationsare converted to Man₅GlcNAc₂ (structural formula IV; FIG. 4A),Man₆GlcNAc₂ (structural formula V; FIG. 4A) and Man₇GlcNAc₂ (structuralformula VI; FIG. 4A). The expression of the other mutant or the wildtype gene cause an altered N-glycosylation phenotype. To evaluatewhether all mutants are equally well expressed, a Western blot analysiswas performed using an antibody specific for an E-tag (a 13 amino acidepitope added to the MNS1 proteins). All mutant proteins, as well as thewild-type MNS1 protein, were expressed equally well.

Example 4 Increasing Phosphorylation Expression of Yarrowia lipolyticaMNN4

To increase the phosphorylation of Man₈GlcNAc₂ , Yarrowia lipolyticaMNN4 (a homologue of the P. pastoris PN01) was overexpressed in Yarrowialipolytica to promote the core type phosphorylation of N-glycans.

The coding sequence of the Yarrowia hPolytica MNN4 (XM_(—)503217,YALI0D24101g) gene was amplified using primersGGCGGATCCATGGTGCTGCACCCGTTTC (YlMNN4 BamHI fw; SEQ ID NO:34) andGGCCCTAGGCTACTCAAACTCCTCGCGAATC (YlMNN4 AvrII rv; SEQ ID NO:35). Thisopen reading frame (ORF) was cloned into the plasmid using BamHI andAvrII sites, which placed the ORF under control of the hp4d promoter ofplasmid pYlHURA3 that contains the URA3d1 gene as a selection marker andthe zeta sequences for improving random integration (FIG. 10).

Prior to transformation in the MTLY60 Δoch1 strain, the plasmidcontaining the MNN4 expression cassette was digested either withEco47III for integration in the URA3 locus, PvuI for integration in theMNN4 locus, or RsrII/BstBI for random integration. Transformantstargeted to the URA3 and MNN4 locus were analysed by PCR using a primerin the hp4d promoter and one in the LIP2 terminator. Transformants withrandom integration of the construct were evaluated by Southern blotanalysis.

To evaluate whether manno-phosphorylation was increased we analysedN-glycans derived from secreted glycoproteins after 48 hours culture inYPD medium by DSA-FACE capillary electrophoresis (FIG. 11). The amountof Man₈GlcNAc₂ (structural formula I) was drastically reduced in favourof two structures that migrate faster (compared to Man₈GlcNAc₂(structural formula I; FIG. 4A)) and that are likely to contain one (P)(structural formula X or XI; FIG. 4B) and two (PP) (structural formulaXII; FIG. 4B) phosphate residues, respectively (FIG. 11). Thus, it canbe concluded that the random integrated expression cassettes performbetter than the cassettes integrated in the URA3 locus or the MNN4locus, in that order. The MZ2 exhibited the highest level ofphosphorylation.

Assuming that both peaks derive from the Man₈GlcNAc₂ (structural formulaI; FIG. 4A) peak, the amount of Man₈GlcNAc₂ converted to phosphorylatedglycans was quantitated (Table 3).

TABLE 3 Phosph- N-glycan struct. height area % signal Status StrainΔoch1 M82P 18 302 1,02826 18.91045* (struct. form. XII) M8P 261 525217,88219 (struct. form. X or XI) M8 928 23816 81,08955 81.08955*(struct. form. I) 29370 100 100 Strain MU5 M82P 1319 19736 27,1677381.17283* (struct. form. XII) M8P 2025 39232 54,00509 (struct. form. Xor XI) M8 539 13677 18,82717 18.82717* (struct. form. I) 72645 100 100Strain MZ2 M82P 1182 17662 27,75299 83.11282* (struct. form. XII) M8P1803 35231 55,35984 (struct. form. X or XI) M8 419 10747 16,8871816.88718* (struct. form. I) 63640 100 100 Table 3 Legend: Height andarea refer to the peak height and peak area as determined fromelectropherograms. “% signal” refers to the proportion of each glycan inthe N-glycan mixture. The numbers identified by asterisk depict theproportion of phosphorylated Man₈Gn₂ (top) and the proportion ofnon-phosphorylated Man₈Gn₂ (bottom).

These results indicated that more than 80% of Man₈GlcNAc₂ (structuralformula I; FIG. 4A) that is present in the parent Δoch1 isphosphorylated in the strain that over expresses the YlMNN4 gene.

Example 5 Modifying Glycosylation by Lipid-linked OligosaccharideModification in the Endoplasmic Reticulum Materials and Methods

Strains, culture conditions and reagents. Escherichia coli strainsMC1061 or TOP10 or DH5α were used for the amplification of recombinantplasmid DNA and grown in a thermal shaker at 37° C. in Luria-Broth (LB)medium supplemented with 100 μg/ml of carbenicillin or 50 μg/ml ofkanamycin depending on the plasmids used.

Yarrowia lipolytica MTLY60 (ura3 leu2) strain was used as parent strain.All yeast strains were cultured in a 28° C. incubator. They were grownon YPD medium (2% dextrose, 2% bacto-peptone and 1% yeast extract) orsynthetic dextrose complete (SDC) medium (0.17% YNB w/o amino acids andwithout ammonium sulphate, 1% glucose, 0.5% NH₄Cl, 50 mM K/Na phosphatebuffer pH 6.8 and 0.077% Complete Supplement Mixture (Qbiogene Inc,Morgan Irvine, Calif.)). For selection of Ura+ and Leu+transformants0.077% CSM −ura or CSM −leu was added respectively.

Standard genetic techniques. Transformation competent cells of Yarrowialipolytica were prepared as described in Boisrame et al. (1996) J. Biol.Chem. 271(20):11668-75, the disclosure of which is incorporated hereinby reference in its entirety. Genomic DNA from all yeast strains wasisolated using a published protocol (Epicenter Kit catologue No.MPY80200; Epicenter Biotechnologies, Madison, Wis.). The protocolinvolves non-enzymatic cell lysis at 65° C., followed by removal ofprotein by precipitation and nucleic acid precipitation andresuspension. PCR amplification was performed in a final volume of 50 μlcontaining 5 μl of 10× buffer (200 mM Tris-HCl pH8.4 and 500 mM KCl), avariable quantity of MgCl₂, 2.5 μM dNTP, 50 ng of template, 50 μmol ofthe proper primers and 2.5 units of either Taq or Pfu DNA polymerase.Cycling conditions used were as follows: denaturation at 94° C. for 10minutes followed by hot start and 30 cycles of 94° C. for 45 seconds,suitable annealing temperature for 45 seconds and extension at 72° C.for 1 minute per kb followed by 10 min of extension at 72° C. DNAfragments (PCR products or fragments) recovered from gel were purifiedusing NucleoSpin extract II (Macherey-Nagel). DNA sequencing wasperformed by VIB Genetic Service Facility (Antwerp, Belgium).

Vector Construction.

(i) Knock-out (gene-replacement) of the ALG3 gene. The promoter fragment(P) of the ALG3 gene (GenBank® Accession No: XM_(—)503488, Genolevures:YALI0E03190g) was amplified from genomic DNA of the Yarrowia lipolyticaMTLY60 strain by PCR with 5′CAGTGCGGCCGCACTCCCTCTTTTCACTCACTATTG3′ (SEQID NO:48) and 5′CATTACCCTGTTATCCCTACGCTCAGATCCAATTGTTTTGGTGGTC3′ (SEQ IDNO:49) as the forward and reverse primers, respectively, using Taqpolymerase (Invitrogen). The overhanging A nucleotide was removed withT4 DNA polymerase (Fermentas, Ontario, Canada). The terminator fragment(T) of the ALG3 gene was amplified from genomic DNA of the Yarrowialipolytica MTLY60 strain by PCR with5′GTAGGGATAACAGGGTAATGCTCTCAAGGACGGACCAGATGAGACTGTTATCG3′(SEQ ID NO:50)and 5′GACTTTAATTAAACCCTATGTGGCACCTCAACCCACATCTCCCGTC3′ (SEQ ID NO:51)

as the forward and reverse primers, respectively, using the proofreadingPfu DNA polymerase (Fermentas). Because of overlapping primer sequencescontaining an ISceI restriction site, both fragments could be linked byPCR with the P-forward primer and the T-reverse primer. This co-ampliconwas then subcloned in a pCR-2.1 TOPO TA (Invitrogen) vector and thecorrectness of the co-amplicon's sequence was confirmed by sequencing.The co-amplicon was then cloned using the NotI-PacI sites into anintermediate vector.

(ii) Overexpression of the ALG6 gene. The ALG6 ORF (1725 bp) togetherwith the terminator (415 bp downstream) of the ALG6 gene (GenBank®Accession No: XM_(—)502922, Genolevures: YALI0D17028g) were cloned fromgenomic DNA of the Yarrowia lipolytica MTLY60 strain by PCR with5′CAGTGGATCCATGAACTCTCCTATTTTCACTACCG3′ (SEQ ID NO:52) and5′GACTCCTAGGAAGCTTCCAGGTTACAAGTTGTTAC3′(SEQ ID NO:53) as the forward andreverse primers, respectively, using the proofreading Pfu DNA polymerase(Fermentas). The sequence was cloned in pCR-Blunt II-TOPO (Invitrogen)and the correctness of the ALG6 ORF sequence was confirmed by sequencing(as above). Next, the ALG6 ORF was cloned in a vector (pYLHmA)containing the hp4d promoter via BamHI and AvrII and subsequently clonedin the intermediate vector via the unique restriction sites ClaI andHindIII present in the terminator fragment of ALG3.

(iii) Selection marker cassette. To remove the selectable marker URA3from the host genomic DNA, the Cre-lox recombination system was used,e.g., as described by Fickers et al. ((2003) J. Microbiol. Methods55(3):727-737, the disclosure of which is incorporated herein byreference in its entirety). Upon expression of the Cre recombinase fromthe plasmid pRRQ2 (hp4d-cre, LEU2) (a gift from the Institut National deRecherche Agronomique (INRA)), the marker gets excised by recombinationbetween the two lox sites. In both constructs, with and without the ALG6overexpression cassette, the URA3 selection marker flanked by lox sites,was inserted in the introduced I-SceI site between P and T fragments ofthe vector, resulting in a “PUT” construct.

Preparation of mannoproteins. Yeast strains were grown overnight in 10ml standard YPD medium in 50 ml falcon tubes, rotating at 250 rpm in a28° C. incubator. The cells were then pelleted by centrifugation at 4000rpm at 4° C. The supernatants were removed, and the cells were firstwashed with 2 ml of 0.9% NaCl solution followed by two washes with 2 mlof water and subsequently resuspended in 1.5 ml of 0.02 M sodium citratepH 7 in a microcentrifuge tube. After autoclaving the tubes for 90minutes at 121° C., the tubes were vortexed and the cellular debris waspelleted by centrifugation. The supernatants were collected and themannoproteins were precipitated overnight with 4 volumes of methanol at4° C. with rotary motion. The precipitate was then obtained bycentrifugation of the alcohol precipitated material. The pellets wereallowed to dry and dissolved in 50 μl of water.

Sugar analysis. DNA sequencer-assisted (DSA), fluorophore-assistedcarbohydrate electrophoresis (FACE) was performed with an ABI 3130 DNAsequencer as described by Callewaert et al. (2001; supra). Briefly,glycoproteins were denatured for 1 hour in RCM buffer (8M urea, 360 mMTris pH 8.6 and 3.2 mM EDTA) at 50° C. followed by immobilization on aprewetted PVDF membrane of a IP plate containing 15 μl RCM. Prewettingof the membrane was done with 300 μl MeOH, 3 times washed with 300 μlwater and 50 μl RCM, followed by vacuum removal. The glycoproteins werereduced for 1 hour with 50 μl 0.1M dithiothreitol and washed 3 timeswith 300 μl water. A 30 minute incubation in the dark with 50 μl 0.1Miodoacetic acid was used to carboxymethylate the SH groups, followed by3 washes with 300 μl water. The plates were subsequently incubated for 1hour with 100 μl 1% polyvinylpyrrolidone 360 to saturate the unoccupiedbinding sites on the membrane, again followed by 3 washes with 300 μlwater. Next, the N-glycans were released by 3 hours treatment withpeptide: N-glycosidase F (PNGase F)×U in 50 μl of 10 mM Tris-acetate pH8.3. N-glycans were recuperated and derivatized with the fluorophore8-aminopyrene-1,3,6-trisulfonate (APTS) by reductive amination. This wasaccomplished with an overnight (ON) incubation at 37° C. with 1 μl of1:1 mixture of 20 mM APTS in 1.2M citric acid and 1M NaCNBH₃ in DMSO andquenching by addition of 4 μl water. Excess label was removed by sizefractionation on Sephadex G-10 resin. The remaining labeled N-glycanswere then concentrated by evaporation. The N-glycans of RNase B and anoligomaltose ladder were included as size markers. Data analysis wasperformed using Genemapper® software (Applied Biosystems). Glycosidasedigests on the labeled sugars were performed ON at 37° C. in 100 mMNH₄AC pH5. Additional Jack bean (JB) mannosidase was added after ONdigestion and left for another 24 hours at 37° C.

Disruption of the ALG3 Gene in Yarrowia lipolytica

To disrupt the ALG3 gene, a vector was generated that includes parts ofthe promoter and terminator of ALG3 and has a URA3 selection markercassette and was designated pYLalg3PUT. A NotI and Pad site wereintegrated to linearize the vector and thereby remove the E. colirelated DNA elements. Double homologous recombination at the promoterand terminator site was used to replace ALG3 with the URA3 selectablemarker, which resulted in an alg3::URA3 mutant strain. The knockoutstrategy applied was described by Fickers et al. (2003; supra) and makesuse of the Cre-lox recombination system, that facilitates efficientmarker rescue. Upon integration in the genomic ALG3 contig the Alg3pα-1,6-mannosyltransferase activity should be lost. This was monitored byanalyzing the glycosylation pattern of the mannoproteins of severaltransformants. The N-glycans derived from mannoproteins were analysed byDSA-FACE (capillary electrophoresis) and treated with a selection ofexoglycosidases to reveal the structures. Seven out of 24 transformantsgave a change in glycosylation profile (three of which are depicted inFIG. 13). In all seven transformants, correct integration of theknockout cassette in the genome could be confirmed by PCR. Three mainglycan structures were found by analyzing the profiles: (i) one(structural formula VII; FIG. 4A) that runs at the same size as theMan₅GlcNAc₂ structure of RNase B (the latter being structural formulaIV; FIG. 4A); (ii) one at a distance of one glucose-unit extra; and(iii) one at the distance of two extra glucose-units. (FIG. 13). Theseresults indicate that ALG3 was disrupted in these cells.

Overexpression of α-1,6-mannosyltransferase Alg6p

A strategy was developed in which a constitutively active overexpressioncassette for the first glucosyltransferase, i.e., Alg6p, wasincorporated into the alg3 gene replacement vector. This vector wasdesignated pYLalg3PUT-ALG6. A NotI/PacI fragment of this vector wastransformed into the Yarrowia lipolytica MTLY60 strain. In this way,disruption of ALG3 and overexpression of ALG6 under control of the hp4dpromoter is achieved. Correct integration in the genome was againconfirmed by PCR. DSA-FACE analysis of the N-glycans derived frommannoproteins showed that half of the transformants, i.e., 12 out of 24,exhibited a change in glycosylation pattern comparing to the WT strain.Overexpression of ALG6 led to a mild clonal variation (FIG. 13).

Identification of the N-glycan Structures

To further elucidate the nature of the glycan structures from theexperiments described above, in vitro digests of glycans derived fromthe mannoproteins (as above) were performed with a selection ofexoglycosidases. The mannoprotein glycans were analyzed with thefollowing enzymes: α-1,2-mannosidase; α-mannosidase (JB) and glucosidaseII. Three observed glycanstructures represent Man₅GlcNAc₂ (structuralformula VII; FIG. 4A), GlcMan₅GlcNAc₂ (structural formula VIII; FIG. 4B)and Glc₂Man₅GlcNAc₂ (structural formula IX; FIG. 4B) (FIG. 14). Theseresults indicate that there is very little to no high mannose elongationby α-1,6-mannosyltranferases (e.g., Och1p).

To determine if ALG6 overexpression is necessary for promotingN-glycosylation site-occupancy, Lipase 2 (LIP2) from Yarrowia lipolyticawas expressed in three different strains of Yarrowia: MTLY60,MTLY60Δalg3 and MTLY60Δalg3ALG6. A construct for the Yarrowia lipolyticaLIP2, under control of a TEF constitutive promoter was obtained fromINRA. The expression cassette was transformed to the above-mentionedstrains and the expression of the protein was verified by subjecting thesupernatant prepared from the transformed cells to SDS-PAGE analysis(FIG. 28). The Lip2p protein has 2 glycosylation sites. Lip2p proteinderived from the alg3-deficient (“knockout”) yeast strain was resolvedby SDS-PAGE into three distinct bands that were visualized usingCoomassie blue staining of the gel (FIG. 28). To confirm that all threeforms of protein in the gel were different glycosylation forms of theLip2p protein, Lip2p protein obtained from the alg3-deficient(“knockout”) yeast strain was subject to treatment with PNGase F (anenzyme that removes oligosaccharide residues from glycoproteins) andthen subjected to SDS-PAGE analysis as described above. Treatment of theLip2p protein with PNGase F resulted in a single band (which had thesame molecular weight as non-glycosylated Lip2p) on the gel followingCoomassie blue staining and indicated that all three forms of proteinpreviously observed were different glycosylation forms of the same Lip2pmolecule. The same is true for the Lip2p derived from the alg3ALG6strain. However, the amount of protein in a reduced glycosylation formis decreased. Thus, it can be concluded that overexpression of ALG6 can(at least partially) restore N-glycosylation site-occupancy, which isreduced in the alg3 knockout mutant yeast strain.

Removing Capping Glucose Structures

Next, to eliminate mono (structural formula VIII; FIG. 4B) andbi-glucosylated (structural formula IX; FIG. 4B) Man₅GlcNAc₂ (structuralformula VII; FIG. 4B) structures in vivo, cells were geneticallyengineered to overexpress the α-subunit of the enzyme glucosidase II.The α subunit of glucosidase II of Yarrowia (GenBank® Accession No:XM_(—)500574) and the α subunit of glucosidase II Trypanosoma brucei(GenBank® Accession No: AJ865333) were independently cloned as twostrategies to overexpress the protein. The α subunit of glucosidase IITrypanosoma brucei was chosen since its natural substrate isGlcMan₅GlcNAc₂ (structural formula VIII; FIG. 4B). Both genes werecloned under control of the constitutive hp4d promoter and theirplasmids contain the URA3 marker. These constructs were transformed intoalg3 mutant yeast strains, both with and without ALG6 overexpression.

Oligosaccharides were prepared from secreted proteins derived fromcultured cells containing the constructs and the profile of theoligosaccharides was determined by DSA-FACE analysis. All transformantsgave the same DSA-FACE profile, two different clones of eachglucosidaseII α are depicted in FIG. 29. From these results it wasconcluded that the overexpression of either the Yarrowia or theTrypanosoma glucosidase II α subunit has only a minor effect on theamount of mono (structural formula VIII; FIG. 4B) and bi-glucosylated(structural formula IX; FIG. 4B) Man₅GlcNAc₂ (structural formula VII;FIG. 4A) structures.

Expression of Glucosidase II α-Subunits of Yarrowia lipolytica andTrypanosoma brucei Tagged with an HDEL Sequence

To improve the effect of the expression of Yarrowia or the Trypanosomaglucosidase II α subunit on removing glucose residues from Man₅GlcNAc₂in vivo, a nucleic acid encoding an HDEL tag was added using molecularbiology techniques in frame to the 3′ end of the nucleic acid encodingeach of the two GlsII α enzymes. The HDEL tag was meant to serve as aretrieval mechanism from the Golgi to the ER. Plasmids encodingHDEL-tagged glucosidase II sequences from both Yarrowia lipolytica(Y.l.) and Trypanosoma brucei (T.b.) under control of the hp4d promoterwere transformed to the alg3 KO strain with and without overexpressionof the ALG6 gene. As can be seen in FIG. 30, overexpression of theYarrowia lipolytica glucosidase II α subunit had only a minor effect onthe amount of glucosylated structures. In contrast, overexpressing theα-Glucosidase II of Typanosoma brucei α subunit with an extra HDEL tagleads to a reduction of the mono-glucose peak (see FIG. 31).

Treatment of Glucosylated Glycans with Mutanase

The above-described results demonstrate one exemplary means of reducingmono-glucosylated forms of Man₅GlcNAc₂. To reduce bi-glucosylated formsof Man₅GlcNAc₂ from glycoproteins, the mutanase of T. harzianum wasinvestigated as one potential solution. An enzyme preparation wasobtained from Novozymes (Novozyme 234; Bagsvaerd, Denmark) and was usedto digest oligosaccharides in vitro. That is, mutanase was added indifferent concentrations to the oligosaccharides derived from a alg3ALG6strain (glycans: Man₅GlcNAc₂, GlcMan₅GlcNAc₂ and Glc₂Man₅GlcNAc₂). Asshown in the DSA-FACE profile of FIG. 32, the bi-glucose peak observedin the oligosaccharides was effectively reduced.

Next, the mutanase of T. harzianum was overexpressed in vivo. AnHDEL-sequence containing mutanase was synthesized as a codon-optimizedcDNA for expression in Yarrowia lipolytica. The mature protein wascloned in frame with the LIP2 pre signal sequence under control of theTEF1 promoter (FIG. 33). This construct is transformed into alg3 mutantyeast strains, both with and without ALG6 overexpression.Oligosaccharides are prepared from cultured cells containing theconstruct and the profile of the oligosaccharides is determined byDSA-FACE analysis. It is expected that the DSA-FACE profile will show areduction in the bi-glucose peak observed in the oligosaccharides. Fromthese results it will be concluded that the overexpression of mutanasein vivo is effective at reducing the bi-glucose peak observed inoligosaccharides as compared to cells not overexpressing the mutanase.

Co-expression of Yl GlsII α- and β Subunits

It is known that the α- and β-subunits of glucosidase II form aheterodimeric complex whereby the β-subunit is responsible for retrievalof the complex to the ER and is also involved in substrate recognition,whereas the α-subunit contains the catalytic activity. Since theoverexpression of only the α-subunit of glucosidase II had a smalleffect on bi-glucose oligosaccharide structures, the α- and β-subunitswere co-expressed.

The open reading frame of the β-subunit (YALI0B03652g) was amplifiedfrom genomic DNA that was isolated from the MTLY60 strain using PCR andwas cloned under control of the TEF1 and hp4d promoter. The constructswere made with LEU2 as a selection marker and with the glucosidase IIβ-subunit under control of the TEF1 and the hp4d promoter. These weretransformed to the alg3 knockout strains with and without ALG6overexpression and overexpressing the Yarrowia lipolytica Glucosidase IIα subunit with and without an HDEL sequence tag. N-glycans were preparedfrom proteins secreted from the cells and the DSA-FACE profiles of theN-glycans are depicted in FIGS. 33 and 34 (alg3 knockout withoverexpression of ALG6). It can be concluded from these profiles thatoverexpressing the β subunit of glucosidase II from Yarrowia lipolyticadid have a positive effect on the trimming of the glucosylated sugars.In general, the efficacy of the β subunit of glucosidase II was improvedwhen expressed under the TEF1 promoter. The glucosylated structures wereeven more reduced when the Yarrowia lipolytica glucosidase II α subunitcontained an HDEL tag (FIGS. 33 and 34).

For alg3-deficient cells without ALG6 overexpression, similar resultsregarding reduction of glucosylated structures were observed for each ofthe different cell populations (FIG. 35).

Expression of Aspergillus GlsII a and b Subunit

In order for the glucose residues to be removed from the glucose bearingstructures that occur in alg3-deficient background, the Aspergillusniger mature (lacking signal peptide) glucosidase II α and β weresynthesized as codon-optimized cDNA for expression in Yarrowialipolytica (α-subunit (SEQ ID NO:7; FIGS. 36A-36B) β-subunit: (SEQ IDNO:8; FIG. 37). Aspergillus niger (An) glucosidase α subunit was clonedunder control of the constitutive TEF1 and hp4d promoters and had URA3gene as a selection marker. The expression cassettes (ORFs under controlof TEF1 and hp4d) were transformed to Yarrowia lipolytica alg3ALG6strain. Transformant candidates were grown in YPD and glycans fromsecreted proteins were analysed by DSA-FACE. It can be deduced from FIG.38 that the two glucosylated structures are less abundant in thetransformant strains compared to the non-transformant (alg3ALG6).

To further reduce the glucosylated glycan structures a construct is madewith β-subunit of the Aspergillus niger glucosidase II under control ofTEF1 promoter or hp4d promoter with LEU2 as a selection marker. Thisconstruct is transformed to Yarrowia lipolytica alg3ALG6 strainexpressing the An glucosidase II α-subunit. It is expected thatexpression of the β-subunit of the Aspergillus niger glucosidase II willresult in a decrease in glucosylated structures in Yarrowia lipolyticacells.

Example 6 Identification of the HAC1 Intron and Cloning and Isolation ofthe HAC1 Gene

Y. lipolytica HAC1 splice site. On the basis of sequence homologybetween the intronic regions of HAC1 in Yarrowia lipolytica and thefungi Trichoderma reesei and Aspergillus nidulans, a potential splicesite of the Yarrowia lipolytica HAC1 (Genbank: XM_(—)500811,Genolevures: Yali0B12716g) was identified. The 5′ and 3′ splice siteswere predicted to be localized in a characteristic loop structure andthe intron was calculated to be 29 bp long.

Primers were developed around the splice site in order to identify theintron. First strand cDNA was synthesized from the isolated mRNA from anUPR (unfolded protein response) induced (by means of growth indithiothreitol (DTT)) and non-induced culture (negative control) withgene specific primers. PCR was then performed on first strand usingprimers HAC1FW06-003 and HAC1Rv06-001. Amplification products wereanalyzed on a 1.5% agarose gel.

A fragment of +/−400 bp was expected to be amplified for the non-inducedcells; a 29 bp smaller fragment was expected to be amplified for theinduced cells. Fragments of the correct size were obtained from thenon-induced cells and the UPR induced cells. Two more amplificationproducts were obtained for the UPR induced culture. The middle fragmentwas the same size as the band obtained for the non-induced culture andwas interpreted as being unspliced HAC1. The lower, most prominent bandwas purified from the gel and cloned into a sequencing vector. Aftersequencing the construct, a sequence alignment was performed in order toidentify the splice site (FIG. 15). From the sequence alignment it canbe seen that the splice site is located at the position that waspredicted from the comparison of the Yarrowia lipolytica and the fungal(Trichoderma reesei and Aspergillus nidulans) HAC1 sequences. The splicesite is 29 bp long.

In order to isolate the active full length HAC1 sequence, primers wereengineered to have restriction sites suitable for cloning into anexpression vector. Primer sequences were as follows: Hac1ylRv07-018:CCTAGGTCACTCCAATCCCCCAAACAGGTTGCTGACGCTCGACTCATAGTGAGCTAGATCAGCAATAAAGTCG(SEQ ID NO:54) and HAC1Fw06-002:GGA TCC ATG TCT ATC AAG CGA GAA GAG TCC(SEQ ID NO:55). A 10 ml culture of yeast cells was incubated for 1.5hours in the presence of 5 mM DTT to induce the UPR response. Followingthe incubation, RNA was isolated from the DTT-treated cells and firststrand cDNA was prepared from the isolated RNA using reversetranscriptase and PCR using the cDNA as a template and the aboveprimers. The PCR-amplified sequence containing the spliced HAC1 wasinserted into the pCR-blunt-TOPO cloning vector using standard molecularbiology techniques and sequenced.

Pichia pastoris HAC1 splice site. On the basis of sequence homology ofthe intronic regions of the Pichia pastoris and Saccharomyces cerevisiaeHAC1 genes, a potential splice site in the Pichia pastoris HAC1 gene wasidentified (FIG. 16). The 5′ and 3′ splice sites were predicted to belocalized in a characteristic loop structure and the intron wascalculated to be 322 bp in length.

Primers (HAC1Fw06-004 and HAC1Rv06-005) were developed around thepredicted splice site in order to identify the intron (see Table 4). Afragment of 257 nucleotides was expected to be amplified when the intronis removed and a 579 bp fragment if intron is still present. Firststrand cDNA was synthesized from the isolated mRNA from an UPR inducedand non-induced culture. The UPR was induced by adding 5 mM DTT to a 10ml culture of exponentially growing cells. The cells were cultured inthe presence of DTT for 1.5 hours. The amplification product wasanalyzed by 1.5% agarose gel electrophoresis. A fragment ofapproximately 257 bp was obtained from cDNA from both non-induced andinduced cells.

TABLE 4 Primers Primer code sequence 5′-->3′ Information HAC1-KarlGAATTCATGCCCGTAGATTC Forward primer Hac1 gene + startTTCTC (SEQ ID NO: 56) codon and EcoRI site HAC1Fw06-004GAGTCTTCCGGAGGATTCA Forward primer Hac1 gene region  G (SEQ ID NO: 57)around 5′ splice site HAC1Rv06-005 CCTGGAAGAATACAAAGTCReverse primer Hac1 gene  (SEQ ID NO: 58) near stop codon HAC1Rv06-009CCTAGGCTATTCCTGGAAG reverse primer Hac1 gene + AATACAAAGTC (SEQ IDstop codon and NO: 59) Avrll site ACTppFw07-007 GGTATTGCTGAGCGTATGCAct1 forward primer for QPCR AAA (SEQ ID NO: 60) ACTppRv07-003CCACCGATCCATACGGAGT Act1 reverse primer for QPCR ACT (SEQ ID NO: 61)HAC1ppFw07-008 CGACCTGGAATCTGCACTT Hac1 forward primer QPCRCAA (SEQ ID NO: 62) HAC1ppRV07-004 CGGTACCACCTAAGGCTTCHac1 reverse primer QPCR CAA (SEQ ID NO: 63) Kar2ppFw07-009CCAGCCAACTGTGTTGATTC Kar2 forward primer QPCR AA (SEQ ID NO: 64)Kar2ppRv07-005 GGAGCTGGTGGAATACCAG Kar2 reverse primer QPCRTCA (SEQ ID NO: 65)

To verify the length of the unspliced P. pastoris HAC1 gene, PCR wasperformed on genomic DNA using primers HAC1-Karl and HAC1Rv06-005. Thelength of the obtained fragment was compared with the length of a PCRproduct obtained from the cDNA from an induced cell culture. Theamplified fragment from the genomic DNA is about 300 bp longer than theamplicon derived from the cDNA using the same primers indicating thatthe intron is present in the genomic DNA sequence and absent from thespliced mRNA.

The cDNA fragment of 257 bp was isolated from the gel and cloned in asequencing vector. The fragment was sequenced and an alignment wasperformed in order to identify the splice site (FIG. 17). To isolate andclone the spliced P. pastoris HAC1 gene, PCR primers were developed withrestriction enzyme sites for cloning into an expression vector(HAC1-Karl and HAC1Rv06-009). A 10 ml culture was UPR-induced with 5 mMDTT for 1.5 hours. First strand cDNA was prepared from the isolated RNAusing reverse transcriptase and PCR was subsequently performed on thecDNA template DNA using the above primers. The spliced HAC1 was isolatedand cloned in pCR-blunt-TOPO cloning vector for sequencing. The splicedgene was also cloned under the control of the methanol inducible AOX1promoter in the expression vector pBLHIS IX to obtain the vector pBLHISIX ppHAC1spliced. The correct insertion of the HAC1 gene into theexpression vector was confirmed using PCR and restriction enzymeanalysis.

In Saccharomyces cerevisiae, upon splicing, the coding sequence of theC-terminal 10 amino acids in the non-spliced mRNA is replaced with thecoding sequence of 18 amino acids. In accordance, in Pichia pastoris itwas revealed that the coding sequence of the C-terminal 45 amino acidsin the non-spliced HAC1 are replaced upon splicing by the codingsequence of again 18 amino acids which are homologous to the ones fromthe S. cerevisiae sequence (FIG. 18).

Example 7 Transformation and Induction of Spliced HAC1 Gene intoYarrowia lipolytica

Yarrowia lipolytica cells (MTLY60 strain) were transformed with thevector “PYHMAXHAC1ylspliced” containing the spliced HAC1 cDNA (above)under the expression control of the hp4d promoter and the URA3 gene as aselection marker. Integration of the vector into the yeast genome wasverified using PCR. The MTLY60 strain transformed withPYHMAXHAC1ylspliced was grown in a 2 ml culture in YPG at 28° C. for 24hours. The cultured cells were washed twice with YNB, then diluted toOD₆₀₀ 0.6 and grown for 24 hours in YTG buffered with 50 mM phosphatebuffer pH: 6.8. The cells were then diluted to OD₆₀₀ 0.2 and grown for 3more generations in order to harvest the cells in the mid-exponentialphase. To the pellet, 1 ml of RNApure™ solution was added to the cellsalong with 1 g of glass beads. Cells were broken by vigorous shaking RNAwas extracted from the broken cells by adding 150 μl chloroform andprecipitating the RNA with isopropanol. The extracted RNA was alsotreated with DNAse to remove any coprecipitated DNA impurities.

First strand cDNA was prepared from 800 ng of the RNA using theiScriptTMcDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, Calif.) ina 20 μl total volume reaction. The equivalent of 20 ng RNA was used forreal time PCR analysis to determine the amount of HAC1 mRNA in thecells. Real time PCR was run using SYBR® green as the detection reagent(fluorescent) (Eurogentec). In addition to designing primers fordetecting the amount of HAC1 mRNA in the cells, primers were alsodesigned to quantify the amount of ACT1 (household gene) and KAR2 (UPRresponsive gene) genes as controls for the real time PCR. The relativeamount of mRNA of each gene in the cells was calculated from thecomparative threshold cycle values using Actin (a housekeeping gene) asthe expression control. Induction of the UPR response by the cells wasconfirmed by measuring the expression of UPR. The expression levels ofKAR2 as well as HAC1 are higher in the strains expressing HAC1 undercontrol of a constitutive promoter compared to the wild type strainMTLY60 (FIG. 39).

Example 8 Transformation and Induction of Spliced HAC1 Gene into Pichiapastoris

Media: For the following experiments, three types of media were used:BMY (Buffered Medium for Yeast: 100 mM potassium phosphate pH: 6.0/1.34%YNB without amino acids/1% Yeast extract/2% peptone); BGMY (BufferedGlycerol-complex Medium for Yeast: 100 mM potassium phosphate pH:6.0/1.34% YNB without amino acids/1% Yeast extract/2% peptone/1%glycerol); and

BMMY (Buffered Methanol-complex Medium for Yeast: 100 mM potassiumphosphate pH: 6.0/1.34% YNB without amino acids/1% Yeast extract/2%peptone/0.5% glycerol).

Pichia pastoris cells were transformed according to the electroporationprotocol from the Pichia Expression kit (Invitrogen Cat. No. K1710-01).The vector pBLHIS IX ppHAC1 spliced was linearized in the HIS4 gene totarget the construct to the HIS4 locus for integration. Ten microgramsof DNA was transformed into the yeast cells. The correct integration ofthe construct was validated using PCR on genomic DNA after isolation ofsingle colonies (primers HAC1-Karl and HAC1Rv06-005). Fragments of 915kb and 1237 kb were amplified from DNA obtained from the transformedcells, whereas in the non-transformants (cells without integration ofthe construct) a fragment of 1237 kb was amplified. Clones so identifiedas positive for integration of the plasmid were grown in 10 ml BMGYmedium for 24 hours before induction. Cells were washed once with BMY.BMGY was added to non-induced cultures while BMY was added to theinduced cultures. Every 12 hours, induced cultures were fed with 0.5%methanol (final concentration). Induction was performed for 24 hoursafter which cells were harvested by centrifugation. To prepare RNA,cells were combined with 1 ml RNApure™ (Genhunter Corporation,Nashville, N.Y.) and 1 g of glass beads, and lysed by vigorous shakingRNA was extracted by the addition of 150 μl chloroform and precipitatedwith isopropanol. The extracted and precipitated RNA was DNAse treatedwith RNAse-free DNAse obtained from Qiagen (Cat No. 79254). 400 ng oftotal RNA was subjected to reverse transcriptase reaction using anoligodT primer and the Superscript II reverse transcriptase (Invitrogen,Cat. No. 18064-014). The equivalent of 20 ng RNA was used in a real-timePCR reaction. Primer sequences were designed by Primer Express software(Applied Biosystems) (see primer table for sequence). Real time PCRutilizing SYBR green fluorescent reagent (Eurogentec) was run in theiCycler machine from BioRad. The relative amounts of mRNA werecalculated from the comparative threshold cycle values using thehousekeeping gene actin as a control. Quantification of UPR is performedthrough expression analysis of the UPR-target gene KAR2. A 3 to 7 foldhigher expression of KAR2 was obtained when comparing clones that werenot induced as compared to the same clones that were induced withmethanol (FIG. 19).

The relative amount of HAC1 mRNA from two additional clones 6 and clone8 was determined by quantitative PCR and compared with the relativeamount of mRNA of Kar2. A strong induction of HAC1 was observed in bothclones. The relative amount of KAR2 mRNA appeared to correlate with therelative amount of HAC1 mRNA, higher expression levels of HAC1 lead tohigher expression level of KAR2 (FIG. 20).

Cell death studies of the methanol-induced cultures were performed usingfluorescence flow cytometry (FFC) and compared to cell death ofnon-induced cultures. Ten thousand cells were measured per analysis.Cells were analyzed on the FACScalibur™ (Becton Dickinson) after 12, 36and 48 hours of induction. No cell death was observed. The GlycoSwitchM5(GSM5) strain has as main core type glycan structures mainly Man₅GlcNAc₂(structural formula IV; FIG. 4). In order to check if Hac1p inductionhas an influence on the N-glycan structure a DSA-FACE analysis wasperformed of 1 ml of the culture medium. The glycan profiles obtainedafter 48 hours of induction of spliced Hac1p are similar to the profileof the parental GSM5 strain.

A growth curve was made in order to check if the induction of Hac1pimpairs the growth of P. pastoris. No growth defect was seen of theHac1p induced strain compared to the empty vector transformed strain(FIG. 22).

Example 9 Expression of YlMNN6

In S. cerevisiae, MNN6 transfers phosphomannose residues to N-glycans.Therefore, overexpression of YlMNN6 in Y. lipolytica could lead toincreased phosphorylation. Moreover, an additional effect onphosphorylation Y. lipolytica be obtained by over expressing YlMNN4 andYlMNN6. The YlMNN6 coding region (Genbank® Accession No. XM_(—)499811,Genolevures Ref: YALI0A06589g) was PCR amplified from the genome usingPCR primers YlMNN6 BamHI fw (GCGGGATCCATGCACAACGTGCACGAAGC (SEQ IDNO:34)) and YlMNN6 AvrII rv (GCGCCTAGGCTACCAGTCACTATAGTTCTCC (SEQ IDNO:35)) and cloned in the pYHmAX expression vector for expression undercontrol of the hp4d promoter (FIG. 21). The plasmid was transformed tothe Y. lipolytica strain MTLY60 using zeta sequences to improve randomintegration. Secreted glycoproteins were collected from cell clones thatgrew on medium without uracil and the composition of the glycanssynthesized the glycoproteins was analyzed using DSA-FACE. However, noincreased phosphorylation was observed (FIG. 22).

Example 10 Effects of Hac1p Expression

Evaluation of Hac1p overexpression on the secretion of heterologousproteins. Vectors containing the hygromycin resistance marker and thespliced HAC1 cDNA under control of the inducible AOX1 promoter(pPIChygppHAC1spliced) or under control of the constitutive GAP promoter(pGAPhygHAC1 ppspliced) were transformed to a GS115 strain expressing amIL-10 protein under the control of the inducible AOX1 promoter. P.pastoris cells were transformed according to the electroporationprotocol from the Pichia Expression kit (Invitrogen Cat. No. K1710-01,Invitrogen, Carlsbad, Calif.). The vectors were linearized in the AOX1or GAP promoter to target the integration of the Hac1p gene torespectively the AOX1 or GAP locus. Integration of the plasmid into thehost genome was confirmed using PCR.

Precultures (5 ml) from positive identified clones were grown in YPD for24 hours. The concentration (OD) at a wavelength of 600 nm (OD₆₀₀) ofthe cells in the cultures was measured and cultures were diluted to anOD₆₀₀ of 1 in 2 ml of BMGY media in each well of a 24 well plate.Cultures were grown in BMGY for 48 hours, washed twice with BMY, andthen induced for 24 hours in BMMY. Every 8 to 12 hours, cultures werere-fed with medium containing 1% methanol (final concentration). Afterinduction, the supernatant of the cells was harvested and the proteinfrom 1 ml of the supernatant was precipitated using trichloroacetic acid(TCA). The precipitated protein was subjected to 15% SDS-PAGE.

From the SDS-PAGE, clonal variation in the expression of at least oneprotein—mIL-10—was observed between the different clones. For example,for the clones expressing the Hac1p protein constitutively (undercontrol of GAP promoter), no improvement in expression level wasobserved, whereas for the clones expressing the Hac1p inducibly (AOX1promoter), two clones could be identified that exhibited higherexpression levels of the mIL-10 protein (FIG. 40 and FIG. 41).Expression of mIL-10 by each of the clones was compared to theexpression of mIL-10 produced by a reference GS115 mL-10 expressingstrain.

A new induction was performed for these clones. A preculture grown for24 hours was diluted to OD 1 in 20 ml BMGY in a baffled flask. Cellswere grown for 48 hours in BMGY, washed twice, and then induced in BMMY.Cultures were re-fed with medium containing 1% methanol every 8-12hours. After induction, the supernatant of the cells was harvested andthe protein from 1 ml of the supernatant was precipitated using TCA.Prior to subjecting the precipitated protein to 15% SDS-PAGE, theprotein was treated with PNGase F (or not) to remove all glycosylation(FIG. 41). SDS-PAGE resolved proteins from the supernatant ofHac1p-expressing strains contained a prominent band of 75 kDa, which isnot present in the reference strain. This band was identified by meansof mass spectrometry as being Kar2p, which is the most prominent UPRtarget gene. It could be shown using the cytokine bead array (CBA) thatsimultaneous inducible expression of the Hac1p and the mIL-10 proteincan lead to a 2 fold higher expression of the mIL-10 protein (clone 1,FIG. 41). CBA was performed on endoH treated mIL-10 protein.

Evaluation of Hac1p overexpression on the surface expression ofheterolgous proteins. Vectors containing the hygromycin resistancemarker and the spliced HAC1 cDNA under control of the inducible AOX1promoter (pPIChygppHAC1spliced) or under control of the constitutive GAPpromoter (pGAPhygHAC1ppspliced) were transformed to GlycoswitchMan5strains expressing a mature human interferon-beta/alpha-agglutininfusion protein, a mature mouse interferon gamma/alpha-agglutinin fusionprotein, a mature human erythropoietin/alpha-agglutinin fusion protein,or a fusion protein of alpha-agglutinin and the lectin-like domain ofmouse thrombomodulin, each of which were under the control of theinducible AOX1 promoter. P. pastoris cells were transformed according tothe electroporation protocol from the Pichia Expression kit (InvitrogenCat. No. K1710-01). The vectors were linearized in the AOX1 or GAPpromoter to target the Hac1p gene to respectively the AOX1 or GAP locusfor integration. Integration of the plasmid into the host genome wasconfirmed using PCR.

Precultures (5 ml) from positive identified clones were grown in YPD for24 hours. The OD₆₀₀ was measured and cultures were diluted to OD₆₀₀ of 1in 2 ml BMGY in each well of a 24 well plate. The cultures were grown inBMGY for 24 hours, washed twice with dionized water, and then induced(using culture medium containing 1% methanol) for 24 hours in BMMY.Surface expression was demonstrated by indirect immunostaining with anantibody specific for the V5-epitope, which is fused C-terminally to theV_(H)H coding sequence. After induction, 10⁷ cells in 1 ml PBS (pH 7.2),supplemented with 0.1% bovine serum albumin (PBS/BSA), were incubatedwith 1 μl/ml of the anti-V5 antibody (1 μg/μl; Invitrogen), washed withPBS/BSA, and incubated with 1 μl/ml Alexa fluor 488-labeled goatanti-mouse IgG (1 μg/μl; Molecular Probes). After washing twice withPBS/BSA, the cells were analyzed by flow cytometry (Table 5).

TABLE 5 MFI values determined by flow cytometry Expressed Wild-typePichia + Pichia + Protein Pichia AOX GAP GAP HAC mouse Interferon- 36.619.9 42.8 gamma human EPO 59.5 45.8 66.5 interferon-beta 22.6 12.4 14.4human 95.5 184.1 67.8 thrombomodulin MFI = Mean Fluorescence Intensityobtained from the flow cytometry analysis.

For the strains expressing the Hac1p protein constitutively noimprovement, or very minor differences, could be observed in surfaceexpression levels for all four proteins compared to reference strainsexpressing the surface protein alone. In cells expressing humaninterferon-beta, a significant reduction of surface expression levelswas observed. For the strains overexpressing the inducible Hac1p (Table5) the following could be observed: 1) in the human interferon-gammasurface expressing strain, a 1.8-fold lowering of the surface expressionlevels could be observed compared to the reference strain expressingalone the human interferon-beta a-agglutinin fusion; 2) for the strainsurface-expressing human erythropoietin a-agglutinin fusion protein, a1.3-fold lowering of the surface expression levels could be observedcompared to the reference strain; 3) in the strain surface expressinghuman interferon-beta, no difference of the surface expression levelscould be observed compared to the reference strain; and 4) in the strainsurface expressing mouse thrombomodulin lectin-like domain, an 1.9-foldincrease of the surface expression levels could be observed compared tothe reference strain.

Effect of overexpression of Hac1p on phosholipid synthesis. To determinewhether overexpression of the Hac1p product (produced from the splicedHAC1 cDNA) had an effect of lipid metabolism in P. pastoris, cells weretransformed with the above-described spliced HAC1 cDNA and the effect ofHac1p on lipid metabolism in the cells was determined by electronmicroscopy analysis. Cells were grown for 48 hours on BMGY, washed oncewith PBS, and then grown for another 48 hours on BMMY. The cells werenext cultured in medium containing 1% methanol every 8 to 12 hours. Thecells were then prepared for electron microscopy according to the methodof Baharaeen (Baharaeen et al. (2004) Mycopathologia). Briefly, aprimary fixative containing glutaraldehyde (3%) and para-formaldehyde(1.5% buffered in 0.05 M sodium cacodylate at pH 7.2) was contacted withthe cells for 2 hours on ice. The cells were then washed three times for20 minutes with 0.05 M sodium cacodylate. After washing, the cells werecontacted with a 6% potassium permanganate solution for one hour at roomtemperature and then washed with 0.05 M sodium cacodylate three timesfor 20 minutes. The results of the experiment are presented in FIG. 54.Overexpression of the Hac1p product (produced from the spliced HAC1cDNA) in P. pastoris lead to the formation of discrete regions ofstacked membranes as can be shown in the electron micrograph (EM)depicted in FIG. 54. These results demonstrate that overexpression ofHac1p, by way of its transcriptional activation of genes involved inlipid metabolism, indeed has a strong effect on lipid metabolism in P.pastoris.

Example 11 Expression of ManHDEL

For Man₅GlcNAc₂ to be bound to glycoproteins expressed by the Δoch1strain, an α-1,2-mannosidase can be expressed to cleave Man₈GlcNAc₂ toMan₅GlcNAc₂ (i.e., Golgi type α-1,2-mannosidase activity). Thismannosidase should be targeted to the secretion system. Trichodermareesei α-1,2-mannosidase (Genbank® accession no. AF212153), fused to theS. cerevisiae prepro mating factor and tagged with a HDEL sequence, isable to trim Man₈GlcNAc₂ to Man₅GlcNAc₂ in vivo in Pichia pastoris aswell as in Trichoderma reesei and Aspergillus niger. An expressionconstruct was made to overexpress MFManHDEL (S. cerevisiae α-matingfactor prepro fused to Trichoderma reesei α-1,2-mannosidase tagged withan HDEL sequence) in Y. lypolytica under control of the constitutivehp4d promoter (FIG. 23). The expression cassette was transformed intothe cells after digestion of the plasmid pYHmAXManHDEL with therestriction enzyme NotI, followed by isolation of the desired fragmentusing agarose-gel electrophoresis.

Glycans derived from mannoproteins from the transformed cells wereanalysed using DSA-FACE. Only A minor fraction of Man₈GlcNAc₂ wasconverted to Man₅GlcNAc₂ (FIG. 24). Incomplete conversion of Man₈GlcNAc₂to Man₅GlcNAc₂ could have been due to a non-optimal secretion signal.Therefore, the Saccharomyces cerevisiae secretion signal was replacedwith the secretion signal derived from the well expressed Yarrowialipolytica LIP2 (LIP2pre). The LIP2pre sequence was made by hybridizingthe synthetic oligonucleotides LIP2pre fwGATCCATGAAGCTTTCCACCATCCTCTTCACAGCCTGCGCTACCCTGGCCGCGGTAC (SEQ ID NO:66)and Lip2prepro rvGTACCGGCCGGCCGCTTCTGGAGAACTGCGGCCTCAGAAGGAGTGATGGGGGAAGGGAGGGCGGC (SEQID NO:67) and cloning the DNA into pYLHmA vector (at the BamHI/AvrIIsites) resulting in the following construct: pYLHUdL2pre. The ManHDELcoding sequence was PCR amplified from pGAPZMFManHDEL usingoligonucleotides ManHDEL Eco47III fw (GGCAGCGCTACAAAACGTGGATCTCCCAAC(SEQ ID NO:68)) and ManHDEL AvrII rv (GGCCCTAGGTTACAACTCGTCGTGAGCAAG(SEQ ID NO:69)) and cloned in pYLHUdL2pre. The construction strategy isdepicted in FIGS. 25A and 25B. The expression cassette (withL2preManHDEL under control of the constitutive promoter hp4d) wastransformed to Yarrowia lipolytica Δoch1 strain after digestion of theplasmid with NotI and isolation of the correct fragment (see above).Glycans derived from secreted proteins were analysed via DSA FACE. Someconversion of Man₈GlcNAc₂ to Man₅GlcNAc₂ occurred, but the reaction wasincomplete (Man₈GlcNAc₂ was present as well as intermediate productsMan₇GlcNAc₂ and Man₆GlcNAc₂; FIG. 26).

To further improve the trimming of Man₈GlcNAc₂, Man₇GlcNAc₂, andMan₆GlcNAc₂ to Man₅GlcNAc₂, the Trichoderma reesei α-1,2 mannosidase wascodon optimized for expression in Yarrowia lipolytica (SEQ ID NO:9; FIG.42) and fused to the LIP2 pre signal sequence. This fusion construct wasexpressed under control of 4 different promoters: (i) hp4d, (ii) GAP(SEQ ID NO:10; FIG. 43), (iii) PDX2, and (iv) TEFL. Final expressionplasmids were named pYLHUXdL2preManHDEL (SEQ ID NO: 11; FIGS. 44A-C)pYLGUXdL2preManHDEL (SEQ ID NO:12; FIGS. 45A-C) pYLPUXdL2preManHDEL (SEQID NO:13; FIGS. 46A-C) pYLTUXdL2preManHDEL (SEQ ID NO:14; FIGS. 47A-C).All 4 plasmids were transformed to Yarrowia lipolytica MTLY60 Δoch1strain (described in example 2) after cutting the plasmid with NotI andisolation of the fragment containing the ManHDEL expression cassette.Transformed strains with the ManHDEL under control of the hp4d, GAP andTEF promoter (plasmids pYLHUXdL2preManHDEL, pYLGUXdL2preManHDEL andpYLTUXdL2preManHDEL) were grown in YPD.

Glycans derived from secreted proteins of transformed strains wereanalyzed by DSA FACE. Results are represented in FIG. 48. Alternatively,transformants (including a transformant that had integrated thepYLPUXdL2preManHDEL plasmid) were grown in medium containing oleic acid(protein production conditions) and glycans were analysed via DSA-FACE.Data for one of the vectors, pYLTUXdL2preManHDEL, are presented in FIG.49. As can be concluded from the data, by 48 hours of culture, almostall glycans are converted to Man₅GlcNAc₂.

Example 12 Culturing Conditions for POX2 Promoter Controlled GeneExpression

Cultures were started from a single colony of a fresh plate and grownovernight in 10 mL YPD at 28° C. in a 50 mL tube in an orbital shaker at250 rpm. Next, a 250 mL shake flask containing 22 mL of productionmedium (including 2.5 mL oleic acid emulsion) was inoculated with thepreculture at a final OD600 of 0.2. This culture was incubated at 28° C.in an orbital shaker at 250 rpm. Samples of the culture were taken atvarious time points over a 96 hour culture.

The oleic acid emulsion (20%) was made the method as follows:

-   Add to a sterile 50 ml vessel;-   20 ml sterile water;-   5 ml oleic acid; and-   125 μl Tween 40.

Sonication resulting in the formation of the emulsion was performed forone minute at 75 Hz.

The production medium consisted of the following:

-   1% yeast extract;-   2% trypton;-   1% glucose; and-   50 mM phosphate pH 6.8.

Example 13 Expression of Human Glucocerebrosidase

Human glucocerebrosidase (GLCM, Swiss Prot entry nr: P04062) waschemically synthesized as a codon-optimized cDNA for expression inYarrowia lipolytica (SEQ ID NO:15; FIG. 50).

The coding sequence for the mature protein was fused to the codingsequence of the LIP2 pre signal sequence. This fusion construct wascloned under control of the oleic acid inducible PDX2 promoter. Theresulting plasmid was named pYLPUXL2preGLCM (=pRAN21)). Beforetransformation, the plasmid was digested with NotI and the fragmentcontaining the expression cassette was isolated and transformed toYarrowia lipolytica strain MTLY60, MTLY60Δoch1 (described in Example 2above), and MTLY60Δoch1ManHDEL (described in Example 11). Transformantsobtained in these three strains were grown as described in Example 12.Proteins were precipitated from the supernatant as described above,subjected to SDS-PAGE, and immunoblotted using a rat monoclonalanti-glucocerebrosidase antibody (Alessandrini et al. (2004) J. Invest.Dermatol 23(6):1030-6). An exemplary immunoblot analysis is depicted inFIG. 51. It can be appreciated from FIG. 51 that in a och1 disruptedstrain no smearing occurs (lanes 1, 2, and 3), whereas heterogeneity ofthe protein is seen as a smear in WT cells (lanes 4 and 6). No smearingof protein was observed in protein obtained from a strain of yeastexpressing ManHDEL. These results demonstrate that a more homogeneouspopulation of a target protein can be obtained using the geneticallyengineered Yarrowia lipolytica cells MTLY60Δoch1 and MTLY60Δoch1ManHDEL.

Example 14 Expression of Human Erythropoietin

Human erythropoietin (Epo, Swiss Prot entry nr: P01588) encoding cDNAwas chemically synthesized codon optimized for expression in Yarrowialipolytica (SEQ ID NO:16; FIG. 52). The cDNA coding sequence for themature protein was fused to the coding sequence of the LIP2 pre signalsequence. This fusion construct was cloned under control of the oleicacid inducible PDX2 promoter. The resulting plasmid was namedpYLPUXL2prehuEPO. Before transformation the plasmid was cut NotI and thefragment containing the expression cassette was isolated and transformedto Yarrowia lipolytica strain MTLY60Δoch1 (described in Example 2).Transformant candidates were grown as described in Example 12 andsecreted proteins were analysed by western blot after SDS PAGE using amonoclonal mouse anti human Epo antibody obtained from R&D systems(clone AE7A5). The EPO product obtained from the cells exhibited veryhomogenous glycosylation.

Example 15 Expression of Human α-Galactosidase A

Human α-galactosidase A (AGAL, Swiss Prot entry nr: P06280) encodingcDNA was chemically synthesized as a codon-optimized cDNA for expressionin Yarrowia lipolytica (SEQ ID NO:17; FIG. 53). FIGS. 12A-12H containthe cloning strategy of the galactosidase.

The cDNA coding sequence for the mature protein was fused to the codingsequence of the LIP2 pre signal sequence. This fusion construct wascloned under control of the oleic acid inducible PDX2 promoter. Theresulting plasmid was named pYLPUXL2preaGalase). Before transformationthe plasmid was cut NotI and the fragment containing the expressioncassette was isolated and transformed to Yarrowia lipolytica strainMTLY60 and MTLY60Δoch1MNN4 (described in Example 4). Transformantsobtained in these two strains were grown as described in Example 12.Extracellular proteins obtained from transformants were analyzed byimmunoblot after SDS-PAGE analysis. Two antibodies specific forα-galactosidase A (a chicken polyclonal antibody obtained from Abcam(ab28962) and a rabbit polyclonal antibody obtained from Santa CruzBiotechnology (sc-25823)) were used to detect the expressed humanα-galactosidase A protein.

Example 16 Expression of Mannosidase in WT Yarrowia lipolytica

To determine whether expression of MannosidaseHDEL alone (that is incells containing a functional OCH1 gene) could lead to a more homogenousglycosylation of proteins expressed by fungal cells, an expressioncassette containing a nucleic acid encoding MannosidaseHDEL (see Example11) was transformed into wild-type Yarrowia lipolytica pold cells.Glycans derived from secreted proteins obtained from the cells wereanalysed by DSA-FACE (FIG. 55). The analyzed glycans consisted mainly ofMan₅GlcNAc₂ and a minor part Man₆GlcNAc₂. These results demonstrate thatexpression of MannosidaseHDEL alone, in the absence of any disruption ofthe OCH1 gene, leads to a more homogenous glycosylation of proteinsexpressed by Yarrowia lipolytica.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of producing an altered N-glycosylationform of a target protein, the method comprising: providing a Yarrowialipolytica or an Arxula adeninivorans cell genetically engineered toexpress a protein that promotes phosphorylation of mannosyl moieties ofN-glycans and to comprise a deficiency in Outer CHain elongation (OCH1)activity; and introducing into the cell a nucleic acid encoding a targetprotein, wherein the cell produces the target protein in an alteredN-glycosylation form relative to the N-glycosylation form of the targetprotein produced in a corresponding non-genetically engineered cell, andwherein the protein that promotes phosphorylation of mannosyl moietiesis: (a)(i) a wild-type MNN4 polypeptide; (a)(ii) a deletion variant thatis the polypeptide of (a)(i) but lacks up to twenty amino acid segments,each segment consisting of two amino acids; (a)(iii) a deletion variantthat is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks upto twenty single, non-contiguous amino acids of the polypeptide of(a)(i) or the variant of (a)(ii); or (a)(iv) a substitution variant thatis the polypeptide of (a)(i), the variant of (a)(ii), or the variant of(a)(iii), but with no more than 10 conservative substitutions, whereinthe variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of thephosphorylation-promoting activity of the polypeptide of (a)(i), (b)(i)a wild-type PNO1 polypeptide; (b)(ii) a deletion variant that is thepolypeptide of (b)(i) but lacks up to twenty amino acid segments, eachsegment consisting of two amino acids; (b)(iii) a deletion variant thatis the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up totwenty single, non-contiguous amino acids of the polypeptide of (b)(i)or the variant of (b)(ii); or (b)(iv) a substitution variant that is thepolypeptide of (b)(i), the deletion variant of (b)(ii), or the deletionvariant of (b)(iii), but with no more than 10 conservativesubstitutions, wherein the variants of (b)(ii), (b)(iii), and (b)(iv)have at least 25% of the phosphorylation-promoting activity of thepolypeptide of (b)(i), or (c) a wild-type MNN6 polypeptide.
 2. Themethod of claim 1, further comprising isolating the alteredN-glycosylation form of the target protein.
 3. The method of claim 1,wherein the target protein is an exogenous protein.
 4. The method ofclaim 1, wherein the target protein is an endogenous protein.
 5. Themethod of claim 1, wherein the target protein is a human protein.
 6. Themethod of claim 1, wherein the target protein is a pathogen protein, alysosomal protein, a growth factor, a cytokine, a chemokine, or a fusionprotein.
 7. The method of claim 1, wherein the target protein is aprotein associated with a lysosomal storage disorder (LSD).
 8. Themethod of claim 7, wherein the lysosomal storage disorder is Gaucherdisease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, orFabry disease.
 9. The method of claims 7, wherein the target protein isglucocerebrosidase or galactocerebrosidase.
 10. The method of claim 7,wherein the target protein is selected from the group consisting ofalpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase,beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase,arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase,aspartylglucosaminidase, iduronate-2-sulfatase,alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase,hyaluronidase, alpha-L-mannosidase, alpha- neuraminidase,phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase,thioesterase, cathepsin K, and lipoprotein lipase.
 11. The method ofclaim 7, wherein the target protein is human alpha-galactosidase A. 12.The method of claim 1, wherein the altered N-glycosylation formcomprises one or more N-glycan structures selected from group consistingof Mans₅G1cNAc₂, Man₈G1cNAc₂, and Man₉G1cNAc₂.
 13. The method of claim1, wherein the altered glycosylation is Mans₅G1cNAc₂, Man₈G1cNAc₂,Man₉G1cNAc₂.
 14. The method of claim 1, wherein the cell furthercomprises a nucleic acid encoding an alpha-mannosidase.
 15. The methodof claim 14, wherein the alpha-mannosidase is an alpha 1,2mannosidase.16. The method of claim 14, wherein the alpha-mannosidase is mannosidase1 (MNS1).
 17. The method of claim 14, wherein the alpha-mannosidase istargeted to the endoplasmic reticulum.
 18. The method of claim 17,wherein the alpha-mannosidase is a fusion protein comprising analpha-mannosidase polypeptide and an HDEL endoplasmic reticulumretention peptide.
 19. The method of claim 1, further comprisingadditional processing of the glycoprotein.
 20. The method of claim 19,wherein the additional processing comprises enzymatic or chemicaltreatment of the altered N-glycosylation form of the target protein. 21.An isolated Yarrowia lipolytica or Arxula adeninivorans cell geneticallyengineered to express a protein that promotes phosphorylation ofmannosyl moieties of N-glycans and to comprise a deficiency in OuterCHain elongation (OCH1) activity,. wherein the protein that promotesphosphorylation of mannosyl moieties is: (a)(i) a wild-type MNN4polypeptide; (a)(ii) a deletion variant that is the polypeptide of(a)(i) but lacks up to twenty amino acid segments, each segmentconsisting of two amino acids; (a)(iii) a deletion variant that is thepolypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twentysingle, non-contiguous amino acids of the polypeptide of (a)(i) or thevariant of (a)(ii); or (a)(iv) a substitution variant that is thepolypeptide of (a)(i), the variant of (a)(ii), or the variant of(a)(iii), but with no more than 10 conservative substitutions, whereinthe variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of thephosphorylation-promoting activity of the polypeptide of (a)(i), (b)(i)a wild-type PNO1polypeptide; (b)(ii) a deletion variant that is thepolypeptide of (b)(i) but lacks up to twenty amino acid segments, eachsegment consisting of two amino acids; (b)(iii) a deletion variant thatis the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up totwenty single, non-contiguous amino acids of the polypeptide of (b)(i)or the variant of (b)(ii); or (b)(iv) a substitution variant that is thepolypeptide of (b)(i), the deletion variant of (b)(ii), or the deletionvariant of (b)(iii), but with no more than 10 conservativesubstitutions, wherein the variants of (b)(ii), (b)(iii), and (b)(iv)have at least 25% of the phosphorylation-promoting activity of thepolypeptide of (b)(i), or (c) a wild-type MNN6 polypeptide.
 22. The cellof claim 21, said cell further comprising a nucleic acid encoding atarget protein, wherein the cell produces the target protein in analtered N-glycosylation form relative to the N-glycosylation form of thetarget protein produced in a corresponding non-genetically engineeredcell.
 23. The cell of claim 21, said cell further comprising a nucleicacid encoding an alpha-mannosidase.
 24. A method of producing an alteredN-glycosylation form of a target protein, the method comprisingcontacting a target protein with a cell lysate prepared from a Yarrowialipolytica or an Arxula adeninivorans cell genetically engineered toexpress a protein that promotes phosphorylation of mannosyl moieties ofN-glycans and to comprise a deficiency in Outer CHain elongation (OCH1)activity, wherein the contacting of the target protein with the celllysate results in an altered N-glycosylation form of the target proteinrelative to the N-glycosylation form of the target protein produced fromthe cell lysate of a corresponding non-genetically engineered cell, andwherein the protein that promotes phosphorylation of mannosyl moietiesis: (a)(i) a wild-type MNN4 polypeptide; (a)(ii) a deletion variant thatis the polypeptide of (a)(i) but lacks up to twenty amino acid segments,each segment consisting of two amino acids; (a)(iii) a deletion variantthat is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks upto twenty single, non-contiguous amino acids of the polypeptide of(a)(i) or the variant of (a)(ii); or (a)(iv) a substitution variant thatis the polypeptide of (a)(i), the variant of (a)(ii), or the variant of(a)(iii), but with no more than 10 conservative substitutions, whereinthe variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of thephosphorylation-promoting activity of the polypeptide of (a)(i), (b)(i)a wild-type PNO1 polypeptide; (b)(ii) a deletion variant that is thepolypeptide of (b)(i) but lacks up to twenty amino acid segments, eachsegment consisting of two amino acids; (b)(iii) a deletion variant thatis the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up totwenty single, non-contiguous amino acids of the polypeptide of (b)(i)or the variant of (b)(ii); or (b)(iv) a substitution variant that is thepolypeptide of (b)(i), the deletion variant of (b)(ii), or the deletionvariant of (b)(iii), but with no more than 10 conservativesubstitutions, wherein the variants of (b)(ii), (b)(iii), and (b)(iv)have at least 25% of the phosphorylation-promoting activity of thepolypeptide of (b)(i), or (c) a wild-type MNN6 polypeptide.
 25. Themethod of claim 24, wherein the cell further comprises a nucleic acidencoding an alpha-mannosidase.
 26. A substantially pure culture ofYarrowia lipolytica or Arxula adeninivorans cells, a substantial numberof which are genetically engineered to express a protein that promotesphosphorylation of mannosyl moieties of N-glycans and to comprise adeficiency in Outer CHain elongation (OCH1) activity, and wherein theprotein that promotes phosphorylation of mannosyl moieties is: (a)(i) awild-type MNN4 polypeptide; (a)(ii) a deletion variant that is thepolypeptide of (a)(i) but lacks up to twenty amino acid segments, eachsegment consisting of two amino acids; (a)(iii) a deletion variant thatis the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up totwenty single, non-contiguous amino acids of the polypeptide of (a)(i)or the variant of (a)(ii); or (a)(iv) a substitution variant that is thepolypeptide of (a)(i), the variant of (a)(ii), or the variant of(a)(iii), but with no more than 10 conservative substitutions, whereinthe variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of thephosphorylation-promoting activity of the polypeptide of (a)(i), (b)(i)a wild-type PNO1 polypeptide; (b)(ii) a deletion variant that is thepolypeptide of (b)(i) but lacks up to twenty amino acid segments, eachsegment consisting of two amino acids; (b)(iii) a deletion variant thatis the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up totwenty single, non-contiguous amino acids of the polypeptide of (b)(i)or the variant of (b)(ii); or (b)(iv) a substitution variant that is thepolypeptide of (b)(i), the deletion variant of (b)(ii), or the deletionvariant of (b)(iii), but with no more than 10 conservativesubstitutions, wherein the variants of (b)(ii), (b)(iii), and (b)(iv)have at least 25% of the phosphorylation-promoting activity of thepolypeptide of (b)(i), or (c) a wild-type MNN6 polypeptide.
 27. Thesubstantially pure culture of claim 26, said cells further comprising anucleic acid encoding a target protein, wherein the cell produces thetarget protein in an altered N-glycosylation form relative to theN-glycosylation form of the target protein produced in a correspondingnon-genetically engineered cell.
 28. The substantially pure culture ofclaim 26, said cell further comprising a nucleic acid encoding analpha-mannosidase.